Alpha-amylase mutants

ABSTRACT

The invention relates to a variant of a parent Termamyl-like alpha-amylase, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to the parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca 2+  dependency and specific activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/100,407filed May 4, 2011, now U.S. Pat. No. 8,263,382, which is a continuationof U.S. application Ser. No. 11/862,580 filed Sep. 27, 2007, now U.S.Pat. No. 7,960,161, which is a division of U.S. application Ser. No.10/980,759 filed Nov. 3, 2004, now U.S. Pat. No. 7,625,737, which is acontinuation of U.S. application Ser. No. 10/644,187 filed Aug. 20,2003, now abandoned, which is a division of U.S. application Ser. No.10/186,042 filed Jun. 28, 2002, now U.S. Pat. No. 6,642,044, which is adivision of U.S. application Ser. No. 09/672,459 filed Sep. 28, 2000,now U.S. Pat. No. 6,436,888, which is a continuation of U.S. applicationSer. No. 09/182,859 filed Oct. 29, 1998, now U.S. Pat. No. 6,143,708,which is a continuation of international application no. PCT/DK97/00197filed Apr. 30, 1997, which claims priority under 35 U.S.C. 119 of Danishapplication nos. 0515/96, 0712/96, 0775/96, and 1263/96 filed Apr. 30,1996, Jun. 28, 1996, Jul. 11, 1996, and Nov. 8, 1996, respectively, thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, inter alia, to novel variants (mutants)of parent Termamyl-like alpha-amylases, notably variants exhibitingalterations in one or more properties (relative to the parent) which areadvantageous with respect to applications of the variants in, inparticular, industrial starch processing (e.g., starch liquefaction orsaccharification).

BACKGROUND OF THE INVENTION

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1)constitute a group of enzymes which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides, andthere is a very extensive body of patent and scientific literaturerelating to this industrially very important class of enzymes.

Among more recent disclosures relating to alpha-amylases, WO 96/23874provides three-dimensional, X-ray crystal structural data for aTermamyl-like alpha-amylase which consists of the 300 N-terminal aminoacid residues of the B. amyloliquefaciens alpha-amylase comprising theamino acid sequence shown in SEQ ID NO: 4 and amino acids 301-483 of theC-terminal end of the B. licheniformis alpha-amylase comprising theamino acid sequence shown in SEQ ID NO: 2 (the latter being availablecommercially under the tradename Termamyl™), and which is thus closelyrelated to the industrially important Bacillus alpha-amylases (which inthe present context are embraced within the meaning of the term“Termamyl-like alpha-amylases”, and which include, inter alia, the B.licheniformis, B. amyloliquefaciens and B. stearothermophilusalpha-amylases). WO 96/23874 further describes methodology fordesigning, on the basis of an analysis of the structure of a parentTermamyl-like alpha-amylase, variants of the parent Termamyl-likealpha-amylase which exhibit altered properties relative to the parent.

SUMMARY OF THE INVENTION

As indicated above, the present invention relates, inter alia, to novelalpha-amylolytic variants (mutants) of a Termamyl-like alpha-amylase, inparticular variants exhibiting altered properties which are advantageousin connection with the industrial processing of starch (starchliquefaction, saccharification and the like).

Alterations in properties which may be achieved in mutants of theinvention are alterations in, e.g., substrate specificity, substratebinding, substrate cleavage pattern, thermal stability, pH/activityprofile, pH/stability profile [such as increased stability at low (e.g.,pH<6, in particular pH<5) or high (e.g., pH>9) pH values], stabilitytowards oxidation, Ca²⁺ dependency, specific activity, and otherproperties of interest. For instance, the alteration may result in avariant which, as compared to the parent Termamyl-like alpha-amylase,has a reduced Ca²⁺ dependency and/or an altered pH/activity profile.

The invention further relates, inter alia, to DNA constructs encodingvariants of the invention, to methods for preparing variants of theinvention, and to the use of variants of the invention, alone or incombination with other alpha-amylolytic enzymes, in various industrialprocesses, e.g., starch liquefaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence, together with the stop codon TAA,encoding the Bacillus strain NCIB 12512 alpha-amylase described in WO95/26397, together with the amino acid sequence of the encodedalpha-amylase (SEQ ID NOS: 38 and 41).

FIG. 2 is an alignment of the amino acid sequences of four parentTermamyl-like alpha-amylases in the context of the invention:

1: the amino acid sequence of the Bacillus strain NCIB 12512alpha-amylase described in WO 95/26397 (SEQ ID NO: 38);

2: the amino acid sequence of the Bacillus strain NCIB 12513alpha-amylase described in WO 95/26397 (SEQ ID NO: 39);

3: the amino acid sequence of the B. stearothermophilus alpha-amylase asshown in SEQ ID NO: 6;

4: the amino acid sequence of the Bacillus sp. #707 alpha-amylasedescribed by Tsukamoto et al., 1988, Biochem. Biophys. Res. Commun. 151:25-31 (SEQ ID NO: 40).

The numbers on the extreme right of the figure give the running totalnumber of amino acids for each of the sequences in question. Note thatfor the sequence numbered 3 (corresponding to the sequence in SEQ ID NO:6), the alignment results in “gaps” at the positions corresponding toamino acid nos. 1 and 175 in the sequences numbered 1, 2 and 4.

FIG. 3 illustrates a PCR strategy employed in Example 2.

DETAILED DISCLOSURE OF THE INVENTION

The Termamyl-like Alpha-amylase

It is well known that a number of alpha-amylases produced by Bacillusspp. are highly homologous on the amino acid level. For instance, the B.licheniformis alpha-amylase comprising the amino acid sequence shown inSEQ ID NO: 2 (commercially available as Termamyl™) has been found to beabout 89% homologous with the B. amyloliquefaciens alpha-amylasecomprising the amino acid sequence shown in SEQ ID NO: 4 and about 79%homologous with the B. stearothermophilus alpha-amylase comprising theamino acid sequence shown in SEQ ID NO: 6. Further homologousalpha-amylases include an alpha-amylase derived from a strain of theBacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all ofwhich are described in detail in WO 95/26397, and the alpha-amylasedescribed by Tsukamoto et al., 1988, Biochemical and BiophysicalResearch Communications 151: 25-31. Still further homologousalpha-amylases include the alpha-amylase produced by the B.licheniformis strain described in EP 0252666(ATCC 27811), and thealpha-amylases identified in WO 91/00353 and WO 94/18314. Othercommercial Termamyl-like B. licheniformis alpha-amylases are Optitherm™and Takatherm™ (available from Solvay), Maxamyl™ (available fromGist-brocades/Genencor), Spezym AA™ (available from Genencor), andKeistase™ (available from Daiwa).

Because of the substantial homology found between these alpha-amylases,they are considered to belong to the same class of alpha-amylases,namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-likealpha-amylase” is intended to indicate an alpha-amylase which, at theamino acid level, exhibits a substantial homology to Termamyl™, i.e.,the B. licheniformis alpha-amylase having the amino acid sequence shownin SEQ ID NO: 2. In other words, a Termamyl-like alpha-amylase is analpha-amylase which has the amino acid sequence shown in SEQ ID NO: 2, 4or 6, or the amino acid sequence shown in SEQ ID NO: 1 of WO 95/26397(which amino acid sequence is shown in FIG. 1 and FIG. 2) or in SEQ IDNO: 2 of WO 95/26397 (which amino acid sequence is shown in FIG. 2) orin Tsukamoto et al. (1988) (which amino acid sequence is shown in FIG.2) or i) which displays at least 60%, such as at least 70%, e.g., atleast 75%, or at least 80%, e.g., at least 85%, at least 90% or at least95% homology with at least one of said amino acid sequences and/or ii)displays immunological cross-reactivity with an antibody raised againstat least one of said alpha-amylases, and/or iii) is encoded by a DNAsequence which hybridizes to the DNA sequences encoding theabove-specified alpha-amylases which are apparent from SEQ ID NOs: 1, 3and 5 of the present application (which encoding sequences encode theamino acid sequences shown in SEQ ID NOs: 2, 4 and 6, respectively),from SEQ ID NO: 4 of WO 95/26397 (which DNA sequence, together with thestop codon TAA, is shown in FIG. 1 and encodes the amino acid sequenceshown in FIG. 1) and from SEQ ID NO: 5 of WO 95/26397, respectively.

In connection with property i), the “homology” may be determined by useof any conventional algorithm, preferably by use of the GAP program fromthe GCG package version 7.3 (June 1993) using default values for GAPpenalties [Genetic Computer Group (1991) Programme Manual for the GCGPackage, version 7, 575 Science Drive, Madison, Wis., USA 53711].

Property ii) of the alpha-amylase, i.e., the immunological crossreactivity, may be assayed using an antibody raised against, or reactivewith, at least one epitope of the relevant Termamyl-like alpha-amylase.The antibody, which may either be monoclonal or polyclonal, may beproduced by methods known in the art, e.g., as described by Hudson etal., 1989. The immunological cross-reactivity may be determined usingassays known in the art, examples of which are Western Blotting orradial immunodiffusion assay, e.g., as described by Hudson et al., 1989.In this respect, immunological cross-reactivity between thealpha-amylases having the amino acid sequences SEQ ID NOs: 2, 4 and 6,respectively, has been found.

The oligonucleotide probe used in the characterization of theTermamyl-like alpha-amylase in accordance with property iii) above maysuitably be prepared on the basis of the full or partial nucleotide oramino acid sequence of the alpha-amylase in question. Suitableconditions for testing hybridization involve presoaking in 5×SSC andprehybridizing for 1 hour at ˜40° C. in a solution of 20% formamide,5xDenhardt′s solution, 50 mM sodium phosphate, pH 6.8, and 50 microgramsof denatured sonicated calf thymus DNA, followed by hybridization in thesame solution supplemented with 100 micro-M ATP for 18 hours at ˜40° C.,or other methods described by, e.g., Sambrook et al., 1989.

In the present context, “derived from” is intended not only to indicatean alpha-amylase produced or producible by a strain of the organism inquestion, but also an alpha-amylase encoded by a DNA sequence isolatedfrom such strain and produced in a host organism transformed with saidDNA sequence. Finally, the term is intended to indicate an alpha-amylasewhich is encoded by a DNA sequence of synthetic and/or cDNA origin andwhich has the identifying characteristics of the alpha-amylase inquestion. The term is also intended to indicate that the parentalpha-amylase may be a variant of a naturally occurring alpha-amylase,i.e., a variant which is the result of a modification (insertion,substitution, deletion) of one or more amino acid residues of thenaturally occurring alpha-amylase.

Parent Hybrid Alpha-amylases

The parent alpha-amylase may be a hybrid alpha-amylase, i.e., analpha-amylase which comprises a combination of partial amino acidsequences derived from at least two alpha-amylases.

The parent hybrid alpha-amylase may be one which on the basis of aminoacid homology and/or immunological cross-reactivity and/or DNAhybridization (as defined above) can be determined to belong to theTermamyl-like alpha-amylase family. In this case, the hybridalpha-amylase is typically composed of at least one part of aTermamyl-like alpha-amylase and part(s) of one or more otheralpha-amylases selected from Termamyl-like alpha-amylases ornon-Termamyl-like alpha-amylases of microbial (bacterial or fungal)and/or mammalian origin.

Thus, the parent hybrid alpha-amylase may comprise a combination ofpartial amino acid sequences deriving from at least two Termamyl-likealpha-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial alpha-amylase, or from at least oneTermamyl-like and at least one fungal alpha-amylase. The Termamyl-likealpha-amylase from which a partial amino acid sequence derives may,e.g., be any of those specific Termamyl-like alpha-amylase referred toherein.

For instance, the parent alpha-amylase may comprise a C-terminal part ofan alpha-amylase derived from a strain of B. licheniformis, and anN-terminal part of an alpha-amylase derived from a strain of B.amyloliquefaciens or from a strain of B. stearothermophilus. Forinstance, the parent alpha-amylase may comprise at least 430 amino acidresidues of the C-terminal part of the B. licheniformis alpha-amylase,and may, e.g., comprise a) an amino acid segment corresponding to the 37N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 4 and an amino acidsegment corresponding to the 445 C-terminal amino acid residues of theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 2, or b) an amino acid segment corresponding to the 68N-terminal amino acid residues of the B. stearothermophilusalpha-amylase having the amino acid sequence shown in SEQ ID NO: 6 andan amino acid segment corresponding to the 415 C-terminal amino acidresidues of the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 2.

The non-Termamyl-like alpha-amylase may, e.g., be a fungalalpha-amylase, a mammalian or a plant alpha-amylase or a bacterialalpha-amylase (different from a Termamyl-like alpha-amylase). Specificexamples of such alpha-amylases include the Aspergillus oryzae TAKAalpha-amylase, the A. niger acid alpha-amylase, the Bacillus subtilisalpha-amylase, the porcine pancreatic alpha-amylase and a barleyalpha-amylase. All of these alpha-amylases have elucidated structureswhich are markedly different from the structure of a typicalTermamyl-like alpha-amylase as referred to herein.

The fungal alpha-amylases mentioned above, i.e., derived from A. nigerand A. oryzae, are highly homologous on the amino acid level andgenerally considered to belong to the same family of alpha-amylases. Thefungal alpha-amylase derived from Aspergillus oryzae is commerciallyavailable under the tradename Fungamyl™.

Furthermore, when a particular variant of a Termamyl-like alpha-amylase(variant of the invention) is referred to—in a conventional manner—byreference to modification (e.g., deletion or substitution) of specificamino acid residues in the amino acid sequence of a specificTermamyl-like alpha-amylase, it is to be understood that variants ofanother Termamyl-like alpha-amylase modified in the equivalentposition(s) (as determined from the best possible amino acid sequencealignment between the respective amino acid sequences) are encompassedthereby.

A preferred embodiment of a variant of the invention is one derived froma B. licheniformis alpha-amylase (as the parent Termamyl-likealpha-amylase), e.g., one of those referred to above, such as the B.licheniformis alpha-amylase having the amino acid sequence shown in SEQID NO: 2.

Construction of Variants of the Invention

The construction of the variant of interest may be accomplished bycultivating a microorganism comprising a DNA sequence encoding thevariant under conditions which are conducive for producing the variant.The variant may then subsequently be recovered from the resultingculture broth. This is described in detail further below.

Altered Properties of Variants of the Invention

The following discusses the relationship between mutations which may bepresent in variants of the invention, and desirable alterations inproperties (relative to those a parent, Termamyl-like alpha-amylase)which may result therefrom.

Decreased Ca²⁺ Dependency

It is highly desirable to be able to decrease the Ca²⁺ dependency of aTermamyl-like alpha-amylase. Accordingly, one aspect of the inventionrelates to a variant of a parent Termamyl-like alpha-amylase, whichvariant exhibits alpha-amylase activity and has a decreased Ca²⁺dependency as compared to the parent alpha-amylase. Decreased Ca²⁺dependency will in general have the functional consequence that thevariant exhibits a satisfactory amylolytic activity in the presence of alower concentration of calcium ion in the extraneous medium than isnecessary for the parent enzyme. It will further often have theconsequence that the variant is less sensitive than the parent tocalcium ion-depleting conditions such as those obtained in mediacontaining calcium-complexing agents (such as certain detergentbuilders).

Decreased Ca²⁺ dependency of a variant of the invention mayadvantageously be achieved, for example, by increasing the Ca²⁺ bindingaffinity relative to that of the parent Termamyl-like alpha-amylase; inother words the stronger the binding of Ca²⁺ in the enzyme, the lowerthe Ca²⁺ dependency.

It may be mentioned here that WO 96/23874 states that amino acidresidues located within 10 Å from a sodium or calcium ion are believedto be involved in, or of importance for, the Ca²⁺ binding capability ofthe enzyme, and that in this connection the mutation N104D [of the B.licheniformis alpha-amylase having the amino acid sequence shown in SEQID NO: 2, or an equivalent (N to D) mutation of an equivalent positionin another Termamyl-like alpha-amylase] is contemplated to be ofparticular interest with respect to decreasing the Ca²⁺ dependency of aTermamyl-like alpha-amylase.

Other mutations mentioned in WO 96/23874 as being of possible importancein connection with Ca²⁺ dependency include mutations which arecontemplated therein to achieve increased calcium binding (and/orthermostability of the enzyme) via stabilization of the C-domain (asdefined in WO 96/23874) of the three-dimensional structure of aTermamyl-like alpha-amylase via formation, for example, of cysteinebridges or salt bridges. Thus, WO 96/23874 discloses that the C-domainof the B. licheniformis alpha-amylase having the amino acid sequenceshown in SEQ ID NO: 2 may be stabilized by introduction of a cysteinebridge between domain A and domain C (as defined in WO 96/23874) byintroduction of the following mutations: A349C+I479C and/or L346C+I430C.

WO 96/23874 likewise discloses that a salt bridge may be obtained byintroduction of one or more of the following mutations in the B.licheniformis alpha-amylase having the amino acid sequence shown in SEQID NO: 2:

-   N457D,E-   N457D,E+K385R-   F350D,E+I430R,K-   F350D,E+I411R,K    and that the calcium site of Domain C may be stabilized by replacing    the amino acid residues H408 and/or G303 with any other amino acid    residue, in particular by introducing one of the substitutions:-   H408Q,E,N,D and/or G303N,D,Q,E    which are contemplated to provide better calcium binding or    protection from calcium depletion (similar mutations in equivalent    positions of other Termamyl-like alpha-amylases are encompassed    hereby).

Other substitution mutations (relative to B. licheniformisalpha-amylase, SEQ ID NO: 2) which are disclosed in WO 96/23874 as beingof apparent importance, inter alfa, in the context of reducing calciumdependency include the following: R23K, H156Y, A181T, A209V, R214, G310Dand P345 (or equivalent mutations in equivalent positions in anotherTermamyl-like alpha-amylase).

In the context of the present invention, further substitution mutationswhich appear to be of importance, inter alia, in relation to reductionof calcium dependency include the following mutations in Domain B (asdefined in WO 96/23874):

A181E,D,Q,N,V (which appear to result in shielding of the outermost Ca²⁺binding site in the junction region between Domain A and Domain B tosome extent);

I201(bulkier amino acid), e.g., I201W,F,L (which appear to result inslight alterations in the geometry of the region in the immediatevicinity of the Ca²⁺—Na⁺—Ca²⁺ binding site(s) in the junction regionbetween Domain A and Domain B, and in the geometry and/or size of anearby hole/cavity); and

Y203E,Q (which are believed to result in stronger binding of theoutermost Ca²⁺ ion in its binding site in the junction region betweenDomain A and Domain B); (or equivalent mutations in equivalent positionsin another Termamyl-like alpha-amylase).

Altered pH Optimum (Altered pH/Activity Profile)

WO 96/23874 discloses that it is contemplated to be possible to changethe pH optimum of a Termamyl-like alpha-amylase, or the enzymaticactivity thereof at a given pH, by changing the pKa of the active siteresidues, and that this may be achieved, e.g., by changing theelectrostatic interaction or hydrophobic interaction between functionalgroups of amino acid side chains of the amino acid residue to bemodified and of its close surroundings.

In the context of the present invention, it is believed on the basis ofelectrostatic considerations [see, e.g., Gilson, 1995, Current Opinionin Structural Biology 5: 216-223; and Honig and Nicholls, 1995, Science268: 1144-1149; and references given therein] and hygroscopicityconsiderations in relation to the three-dimensional structure of theTermamyl-like alpha-amylase disclosed in WO 96/23874 that mutations ofrelevance, inter alfa, for altering (increasing or decreasing) the pHoptimum of a Termamyl-like alpha-amylase include the following mutationsor equivalents thereof [referring here to the sequence of B.licheniformis alpha-amylase (SEQ ID NO: 2)]:

Q9K,L,E; F11R,K,E; E12Q; D100N,L; V101H,R,K,D,E,F; V102A,T; I103H,K;N104R,K,D; H105R,K,D,E,W,F; L196R,K,D,E,F,Y; I212R,K,D,E; L230H,K,I;A232G,H,F,S,V; V233D; K234L,E; I236R,K,N,H,D,E; L241R,K,D,E,F; A260S;W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E; L270R,K,H,D,E; V283H,D;F284H; D285N,L; V286R,K,H,D,E; Y290R,E; V312R,K,D,E; F323H; D325N;N326K,H,D,L; H327Q,N,E,D,F; Q330L,E; G332D; Q333R,K,H,E,L;S334A,V,T,L,I,D; L335G,A,S,T,N; E336R+R375E; T337D,K; T338D,E; T339D;Q360K,R,E; D365N; G371D,R.Increased Stability at Low (Acidic) pH

In the context of the present invention, mutations (amino acidsubstitutions) of importance with respect to achieving increasedstability at low pH appear to include mutations corresponding to thefollowing mutations in the B. licheniformis alpha-amylase having theamino acid sequence shown in SEQ ID NO: 2:

-   mutations at positions H68, H91, H247, R305, K306, H382, K389, H405,    H406, H450 or R483;-   the mutations:-   H140Y;-   H142Y;-   H156Y;-   H159Y;-   H140D+H142R;-   H140K+H142D; or-   H142Y+H156Y    as well as combinations of any two or more of these mutations.    Increased Thermostability and/or Altered Temperature Optimum    (Altered Temperature/Activity Profile)

A further aspect of the invention relates to a variant of a parentTermamyl-like alpha-amylase, which variant is the result of one or moreamino acid residues having been deleted from, substituted in or added tothe parent alpha-amylase so as to achieve increased thermostability ofthe variant.

In may be mentioned that in relation to achieving increasedthermostability, WO 96/23874 discloses that a particularly interestingvariant of a Termamyl-like alpha-amylase comprises a mutationcorresponding to one of the following mutations (using the numbering ofthe B. licheniformis alpha-amylase amino acid sequence shown in SEQ IDNO: 2):

-   L61W,V,F;-   Y62W;-   F67W;-   K106R,F,W;-   G145F,W-   I212F,L,W,Y,R,K;-   S151 replaced with any other amino acid residue and in particular    with F,W,I or L;-   R214W;-   Y150R,K;-   F143W; and/or-   R146W.

WO 96/23874 further discloses in this connection that the mutationscorresponding to one or more of the following mutations in the B.licheniformis alpha-amylase having the amino acid sequence shown in SEQID NO: 2 are of interest in relation to achieving increasedthermostability relative to that of the parent alpha-amylase:

-   L241I,F,Y,W; and/or-   I236L,F,Y,W-   L7F,I,W-   V259F,I,L-   F284W-   F350W-   F343W-   L427F,L,W-   V481,F,I,L,W.

In the context of the present invention, it can be seen from analignment of the amino acid sequences of alpha-amylases from variousBacillus species that B. licheniformis alpha-amylase and B.amyloliquefaciens alpha-amylase both contain an “insertion” of threeamino acids relative to, e.g., B. stearothermophilus alpha-amylase.

From a model of the structure of B. licheniformis alpha-amylase built onthe basis of the three-dimensional structure of the Termamyl-likealpha-amylase disclosed in WO 96/23784 (vide supra), taking into accountthe homology of B. licheniformis alpha-amylase to the Termamyl-likealpha-amylase in question, it can be seen that the above-mentioned“insertion” lies within a part of the structure denoted “loop 8” in WO96/23784, making this loop bulkier in B. licheniformis alpha-amylasethan in the Termamyl-like alpha-amylase and resulting in a loop thatprotrudes from the structure, thereby possibly destabilizing thestructure. It is therefore contemplated that deletion of one or moreamino acids in the region in question in B. licheniformis or B.amyloliquefaciens alpha-amylase will improve the thermostability ofthese alpha-amylases.

Especially interesting in this connection is deletion of three aminoacids within the partial sequence from T369 to I377 (referring to theamino acid sequence of B. licheniformis alpha-amylase shown in SEQ IDNO: 2), i.e., the partial sequence:T369-K370-G371-D372-S373-Q374-R375-E376-I377 (or the correspondingpartial sequence in B. amyloliquefaciens alpha-amylase). In addition tosuch deletions, substitution of one or more of the undeleted amino acidswithin the latter partial sequence may also be advantageous.

Preferable deletions of three amino acids in the partial sequence fromT369 to I377 (in the B. licheniformis alpha-amylase) are deletion ofK370+G371+D372 (i.e., K370*+G371*+D372* ) or deletion of D372+S373+Q374(i.e., D372*+S373*+Q374* ) (or equivalent deletions in the correspondingpartial sequence in B. amyloliquefaciens alpha-amylase).

Another type of mutation which would appear to be of value in improvingthe thermostability of these alpha-amylases is the substitution(replacement) of the entire partial amino acid sequence from T369 toI377 (referring to the sequence of the B. licheniformis alpha-amylase)with one of the following partial sequences of six amino acids (sequencenumbering increasing from left to right): I-P-T-H-S-V; I-P-T-H-G-V; andI-P-Q-Y-N-I (or one of the same substitutions of the correspondingpartial sequence in B. amyloliquefaciens alpha-amylase).

Other mutations which can apparently be of some importance in relationto achieving increased thermostability include amino acid substitutionsat the following positions (referring to SEQ ID NO: 2):

-   R169 (e.g., R169I,L,F,T);-   R173 (especially R173I,L,F,T);-   I201F;-   I212F;-   A209L,T; or-   V208I    as well as combinations of any two or more of these mutations.

Increased thermostability at acidic pH and/or at low Ca²⁺ concentration

In the context of the invention, mutations which appear to be ofparticular relevance in relation to obtaining variants according to theinvention having increased thermostability at acidic pH (pH<7) and/or atlow Ca²⁺ concentration include mutations at the following positions(relative to B. licheniformis alpha-amylase, SEQ ID NO: 2):

-   H156, N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264,    N265

It may be mentioned here that N and E amino acid residues, respectively,at positions corresponding to N109 and E211, respectively, in SEQ ID NO:2 constitute amino acid residues which are conserved in numerousTermamyl-like alpha-amylases. Thus, for example, the correspondingpositions of these residues in the amino acid sequences of a number ofTermamyl-like alpha-amylases which have already been mentioned (videsupra) are as follows:

Termamyl-like alpha-amylase N position E position B. licheniformis (SEQID NO: 2) N190 E211 B. amyloliquefaciens (SEQ ID NO: 4) N190 E211 B.stearothermophilus (SEQ ID NO: 6) N193 E210 Bacillus NCIB 12512 (WO95/26397) N195 E212 Bacillus NCIB 12513 (WO 95/26397) N195 E212“Bacillus sp. #707” (Tsukamoto et al.) N195 E212

Mutations of these conserved amino acid residues appear to be veryimportant in relation to improving thermostability at acidic pH and/orat low calcium concentration, and the following mutations are ofparticular interest in this connection (with reference to the numberingof the B. licheniformis amino acid sequence shown in SEQ ID NO: 2):

-   H156Y,D-   N172R,H,K-   A181T-   N188P-   N190L,F-   H205C-   D207Y-   A209L,T,V-   A210S-   E211Q-   Q264A,E,L,K,S,T-   N265A,S,T,Y    as well as any combination of two or more of these mutations.

An example of a particularly interesting double mutation in thisconnection is Q264S+N265Y.

Altered Cleavage Pattern

In the starch liquefaction process it is desirable to use analpha-amylase which is capable of degrading the starch molecules intolong, branched oligosaccharides, rather than an alpha-amylase whichgives rise to formation of shorter, branched oligosaccharides (likeconventional Termamyl-like alpha-amylases). Short, branchedoligosaccharides (panose precursors) are not hydrolyzed satisfactorilyby pullulanases, which are used after alpha-amylase treatment in theliquefaction process, but before addition of a saccharifyingamyloglucosidase (glucoamylase). Thus, in the presence of panoseprecursors, the product mixture present after the glucoamylase treatmentcontains a significant proportion of short, branched, so-calledlimit-dextrin, viz. the trisaccharide panose. The presence of panoselowers the saccharification yield significantly and is thus undesirable.

Thus, one aim of the present invention is to arrive at a mutantalpha-amylase having appropriately modified starch-degradationcharacteristics but retaining the thermostability of the parentTermamyl-like alpha-amylase.

It may be mentioned here that according to WO 96/23874, variantscomprising at least one of the following mutations are expected toprevent cleavage close to the branching point:

-   V54L,I,F,Y,W,R,K,H,E,Q-   D53L,I,F,Y,W-   Y56W-   Q333W-   G57 all possible amino acid residues-   A52 amino acid residues larger than A, e.g., A52W,Y,L,F,I.    Increased Specific Activity

In a further aspect of the present invention, important mutations withrespect to obtaining variants exhibiting increased specific activityappear to include mutations corresponding to the following mutations inthe B. licheniformis alpha-amylase having the amino acid sequence shownin SEQ ID NO: 2:

-   mutations (amino acid substitutions) at positions S187 (especially    S187D) or Q264 (e.g., Q264R,K,S);-   mutations (substitutions) at position Y290 (especially Y290E,K);-   the mutation V54I;    as well as combinations of any two or more of the latter mutations,    or combinations of one, two or more of the latter mutations with the    following multiple mutations:-   A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I    General Mutations in Variants of the Invention

It may be preferred that a variant of the invention comprises one ormore modifications in addition to those outlined above. Thus, it may beadvantageous that one or more proline residues present in the part ofthe alpha-amylase variant which is modified is/are replaced with anon-proline residue which may be any of the possible, naturallyoccurring non-proline residues, and which preferably is an alanine,glycine, serine, threonine, valine or leucine.

Analogously, it may be preferred that one or more cysteine residuespresent among the amino acid residues with which the parentalpha-amylase is modified is/are replaced with a non-cysteine residuesuch as serine, alanine, threonine, glycine, valine or leucine.

Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino acid fragment 185-209of SEQ ID NO: 2 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like alpha-amylase, of oneor more of the Lys residues present in an amino acid fragmentcorresponding to the amino acid fragment 185-209 of SEQ ID NO: 2 by anArg.

It will be understood that the present invention encompasses variantsincorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce point-mutations in anyof the variants described herein.

Methods for Preparing Alpha-amylase Variants

Several methods for introducing mutations into genes are known in theart. After a brief discussion of the cloning of alpha-amylase-encodingDNA sequences, methods for generating mutations at specific sites withinthe alpha-amylase-encoding sequence will be discussed.

Cloning a DNA Sequence Encoding an Alpha-amylase

The DNA sequence encoding a parent alpha-amylase may be isolated fromany cell or microorganism producing the alpha-amylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the alpha-amylase to be studied. Then,if the amino acid sequence of the alpha-amylase is known, homologous,labelled oligonucleotide probes may be synthesized and used to identifyalpha-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to a known alpha-amylase gene could beused as a probe to identify alpha-amylase-encoding clones, usinghybridization and washing conditions of lower stringency.

Yet another method for identifying alpha-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming alpha-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for alpha-amylase, thereby allowingclones expressing the alpha-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described by Beaucage and Caruthers (1981) orthe method described by Matthes et al. (1984). In the phosphoroamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or Saikiet al. (1988).

Site-directed Mutagenesis

Once an alpha-amylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al. (1984). U.S. Pat.No. 4,760,025 discloses the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylase-encoding DNAsequences is described in Nelson and Long (1989). It involves the 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Random Mutagenesis

Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence in question, or within the wholegene.

WO 96/23874 discloses that in connection with achieving improved bindingof a substrate (i.e., improved binding of a carbohydrate species, suchas amylose or amylopectin) by a Termamyl-like alpha-amylase variant,modified (e.g., higher) substrate specificity and/or modified (e.g.,higher) specificity with respect to cleavage (hydrolysis) of substrate,the following codon positions for the amino acid sequence shown in SEQID NO: 2 (or equivalent codon positions for another parent Termamyl-likealpha-amylase in the context of the invention) appear to be particularlyappropriate for targetting:

-   13-18-   50-56-   70-76-   102-109-   163-172-   189-199-   229-235-   360-364-   327-335    Improvement of Liquefaction Performance at low pH and Low Calcium    Ion Concentration

For an alpha-amylase to be used in a starch liquefaction process it isof particular interest that it be thermostable and able to function atlow pH and low calcium concentrations. In order to improve theseproperties of a parent Termamyl-like alpha-amylase, in particular the B.licheniformis alpha-amylase or a variant or hybrid thereof, randommutagenesis (preferably by use of doped or spiked oligonucleotideprimers) followed by appropriate selection of the resulting mutatedenzymes may be performed. The direction of selection of regions torandomize and selection of doping are based primarily on stabilizationof calcium ions already present, and on improvement in residue/residueor domain/domain electrostatic interactions at low pH. In addition, theregions which have been shown to include positions important forachieving good starch liquefaction performance may be selected.

In order to prepare a variant of a parent Termamyl-like alpha-amylasehaving the above properties, at least one of the following regions mayadvantageously be subjected to random mutagenesis (the numbering of theamino acid residues being as in SEQ ID NO: 2):

Region Residue Description I: 153-163 Calcium region between domain A &B, also containing H156 II: 178-192 Calcium region between domain A & BIII: 193-214 Calcium region between domain A & B, also containing A209IV: 232-237 Calcium region between domain A & B V: 297-308 Calciumregion between domain A & C VI: 403-409 Calcium region between domain A& C VII: 428-435 Calcium region between domain A & C VIII: 131-136Region containing H133 IX: 164-175 Region in contact with H133 region X:262-278 Region containing Q264

Preferably, two, three or four of the above regions are subjected torandom mutagenesis in the construction of a novel alpha-amylase variantof the invention. For instance, the following combinations of regionsare suitably subjected to random mutagenesis:

-   VIII+IX-   VIII+IX+II-   II+III+IV-   IV+I

Furthermore, it is preferred that the mutagenesis is carried out by useof doped or spiked oligonucleotides. The doping is preferably done so asto introduce amino acids contributing to improved stability at low pHand reduced calcium dependency at low pH of the resulting alpha-amylasevariant. Furthermore, when selecting the doping scheme, the possibilityof introducing Asn and Gln residues should generally be avoided, sinceAsn and Gln residues in general are associated with instability at lowpH. Preferably, when a Pro residue can be inserted with potentialbenefits (e.g., as assessed from protein-structural considerations), thedoping scheme is prepared to include a preference for introduction of aPro residue.

The parent Termamyl-like alpha-amylase to be subjected to randommutagenesis according to the above principle may be any wild typealpha-amylase or a variant thereof containing one or more mutations. Theparent may be a hybrid between at least two alpha-amylases as explainedin further detail herein. Preferably, the parent alpha-amylase is amutant of the B. licheniformis alpha-amylase having the sequence shownin SEQ ID NO: 2 containing at least one mutation, and preferablymultiple mutations. The parent alpha-amylase may alternatively be ahybrid alpha-amylase which contains at least a part of the B.licheniformis (SEQ ID NO: 2) alpha-amylase. Specific examples of parentalpha-amylases suited to mutagenesis according to the above-describedprinciples include: variants of the B. licheniformis (SEQ ID NO: 2)alpha-amylase which contain at least one of, i.e., one, two, three, fouror all five of, the mutations H156Y, A181T, N190F, A209V and Q264S;hybrid alpha-amylases which contain a part of the B. licheniformis (SEQID NO: 2) alpha-amylase, preferably a C-terminal part thereof, such asamino acids 35-483 thereof, and a part of another Termamyl-likealpha-amylase such as B. amyloliquefaciens (SEQ ID NO: 4) alpha-amylase,preferably an N-terminal part thereof such as the first 38 amino acidresidues thereof.

In relation to the above, a further aspect of the present inventionrelates to a method for generating a variant of a parent Termamyl-likealpha-amylase, which variant exhibits increased stability at low pH andat low calcium concentration relative to the parent, the methodcomprising:

(a) subjecting a DNA sequence encoding the parent Termamyl-likealpha-amylase to random mutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a hostcell, and

(c) screening for host cells expressing a mutated alpha-amylase whichhas increased stability at low pH and low calcium concentration relativeto the parent alpha-amylase.

Step (a) of the latter method of the invention is preferably performedusing doped primers, as described in the working examples herein (videinfra).

Method of Performing Random Mutagenesis

The random mutagenesis of a DNA sequence encoding a parent alpha-amylaseto be performed in accordance with step a) of the above-described methodof the invention may conveniently be performed by use of any methodknown in the art.

For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents.

The mutagenizing agent may, e.g., be one which induces transitions,transversions, inversions, scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the DNA sequence encoding the parent enzyme to be mutagenizedin the presence of the mutagenizing agent of choice under suitableconditions for the mutagenesis to take place, and selecting for mutatedDNA having the desired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at the positionswhich are to be changed. The doping or spiking may be done so thatcodons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding the amylolyticenzyme by any published technique, using, e.g., PCR, LCR or any DNApolymerase and ligase.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed to have a preferencefor the introduction of certain nucleotides, and thereby a preferencefor introduction of one or more specific amino acid residues. The dopingmay, e.g., be made so as to allow for the introduction of 90% wild typeand 10% mutations in each position. An additional consideration inchoice of doping scheme is genetic as well as protein-structuralconstraints. The doping scheme may be made by using the DOPE program(see the working examples herein) which, inter alia, ensures thatintroduction of stop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a parent alpha-amylase enzyme is subjected toPCR under conditions that increase the misincorporation of nucleotides(Deshler (1992); Leung et al., 1989, Technique 1: 11-15).

A mutator strain of E. coli (Fowler et al., 1974, Molec. Gen. Genet.133: 179-191), S. cereviseae or any other microbial organism may be usedfor the random mutagenesis of the DNA encoding the amylolytic enzyme by,e.g., transforming a plasmid containing the parent enzyme into themutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mutatedplasmid may subsequently be transformed into the expression organism.

The DNA sequence to be mutagenized may conveniently be present in agenomic or cDNA library prepared from an organism expressing the parentamylolytic enzyme. Alternatively, the DNA sequence may be present on asuitable vector such as a plasmid or a bacteriophage, which as such maybe incubated with or otherwise exposed to the mutagenizing agent. TheDNA to be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to the expression step (b) or the screening step (c) beingperformed. Such amplification may be performed in accordance withmethods known in the art, the presently preferred method beingPCR-generated amplification using oligonucleotide primers prepared onthe basis of the DNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenizing agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich was carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram-positive bacteria such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

Localized random mutagenesis: the random mutagenesis may advantageouslybe localized to a part of the parent alpha-amylase in question. Thismay, e.g., be advantageous when certain regions of the enzyme have beenidentified to be of particular importance for a given property of theenzyme, and when modified are expected to result in a variant havingimproved properties. Such regions may normally be identified when thetertiary structure of the parent enzyme has been elucidated and relatedto the function of the enzyme.

The localized random mutagenesis is conveniently performed by use ofPCR-generated mutagenesis techniques as described above, or any othersuitable technique known in the art.

Alternatively, the DNA sequence encoding the part of the DNA sequence tobe modified may be isolated, e.g., by being inserted into a suitablevector, and said part may subsequently be subjected to mutagenesis byuse of any of the mutagenesis methods discussed above.

With respect to the screening step in the above-mentioned method of theinvention, this may conveniently performed by use of an assay asdescribed in connection with Example 2.

With regard to screening in general, a filter assay based on thefollowing is generally applicable:

A microorganism capable of expressing the mutated amylolytic enzyme ofinterest is incubated on a suitable medium and under suitable conditionsfor the enzyme to be secreted, the medium being provided with a doublefilter comprising a first protein-binding filter and on top of that asecond filter exhibiting a low protein binding capability. Themicroorganism is located on the second filter. Subsequent to theincubation, the first filter comprising enzymes secreted from themicroorganisms is separated from the second filter comprising themicroorganisms. The first filter is subjected to screening for thedesired enzymatic activity and the corresponding microbial coloniespresent on the second filter are identified.

The filter used for binding the enzymatic activity may be any proteinbinding filter, e.g., nylon or nitrocellulose. The topfilter carryingthe colonies of the expression organism may be any filter that has no orlow affinity for binding proteins, e.g., cellulose acetate or Durapore™.The filter may be pretreated with any of the conditions to be used forscreening or may be treated during the detection of enzymatic activity.

The enzymatic activity may be detected by a dye, fluorescence,precipitation, pH indicator, IR-absorbance or any other known techniquefor detection of enzymatic activity.

The detecting compound may be immobilized by any immobilizing agent,e.g., agarose, agar, gelatine, polyacrylamide, starch, filter paper,cloth; or any combination of immobilizing agents.

Alpha-amylase activity is detected by Cibacron Red labelled amylopectin,which is immobilized on agarose. For screening for variants withincreased thermal and high-pH stability, the filter with boundalpha-amylase variants is incubated in a buffer at pH 10.5 and 60 or 65°C. for a specified time, rinsed briefly in deionized water and placed onthe amylopectin-agarose matrix for activity detection. Residual activityis seen as lysis of Cibacron Red by amylopectin degradation. Theconditions are chosen to be such that activity due to the alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 2 can barely bedetected. Stabilized variants show, under the same conditions, increasedcolour intensity due to increased liberation of Cibacron Red.

For screening for variants with an activity optimum at a lowertemperature and/or over a broader temperature range, the filter withbound variants is placed directly on the amylopectin-Cibacron Redsubstrate plate and incubated at the desired temperature (e.g., 4° C.,10° C. or 30° C.) for a specified time. After this time activity due tothe alpha-amylase having the amino acid sequence shown in SEQ ID NO: 2can barely be detected, whereas variants with optimum activity at alower temperature will show increase amylopectin lysis. Prior toincubation onto the amylopectin matrix, incubation in all kinds ofdesired media—e.g., solutions containing Ca²⁺, detergents, EDTA or otherrelevant additives—can be carried out in order to screen for changeddependency or for reaction of the variants in question with suchadditives.

Testing of Variants of the Invention

The testing of variants of the invention may suitably be performed bydetermining the starch-degrading activity of the variant, for instanceby growing host cells transformed with a DNA sequence encoding a varianton a starch-containing agarose plate and identifying starch-degradinghost cells. Further testing as to altered properties (including specificactivity, substrate specificity, cleavage pattern, thermoactivation, pHoptimum, pH dependency, temperature optimum, and any other parameter)may be performed in accordance with methods known in the art.

Expression of Alpha-amylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding analpha-amylase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an alpha-amylase variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensalpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the alpha-amylasevariant of the invention. Termination and polyadenylation sequences maysuitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g., when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. In general, the Bacillusalpha-amylases mentioned herein comprise a preregion permittingsecretion of the expressed protease into the culture medium. Ifdesirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the inventionencoding an alpha-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al. (1989)).

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of an alpha-amylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyceslividans or Streptomyces murinus, or gram-negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favourably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae.The filamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

In a yet further aspect, the present invention relates to a method ofproducing an alpha-amylase variant of the invention, which methodcomprises cultivating a host cell as described above under conditionsconducive to the production of the variant and recovering the variantfrom the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the alpha-amylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

The alpha-amylase variant secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Industrial Applications

The alpha-amylase variants of this invention possess valuable propertiesallowing for a variety of industrial applications. In particular, enzymevariants of the invention are applicable as a component in washing,dishwashing and hard-surface cleaning detergent compositions. Numerousvariants are particularly useful in the production of sweeteners andethanol from starch, and/or for textile desizing. Conditions forconventional starch-conversion processes, including starch liquefactionand/or saccharification processes, are described in, e.g., U.S. Pat. No.3,912,590 and in EP patent publications nos. 252,730 and 63,909.

Production of sweeteners from starch: A “traditional” process forconversion of starch to fructose syrups normally consists of threeconsecutive enzymatic processes, viz. a liquefaction process followed bya saccharification process and an isomerization process. During theliquefaction process, starch is degraded to dextrins by an alpha-amylase(e.g., Termamyl™) at pH values between 5.5 and 6.2 and at temperaturesof 95-160° C. for a period of approximately 2 hours. In order to ensurean optimal enzyme stability under these conditions, 1 mM of calcium isadded (40 ppm free calcium ions).

After the liquefaction process the dextrins are converted into dextroseby addition of a glucoamylase (e.g., AMG™) and a debranching enzyme,such as an isoamylase or a pullulanase (e.g., Promozyme™). Before thisstep the pH is reduced to a value below 4.5, maintaining the hightemperature (above 95° C.), and the liquefying alpha-amylase activity isdenatured. The temperature is lowered to 60° C., and glucoamylase anddebranching enzyme are added. The saccharification process proceeds for24-72 hours.

After the saccharification process the pH is increased to a value in therange of 6-8, preferably pH 7.5, and the calcium is removed by ionexchange. The dextrose syrup is then converted into high fructose syrupusing, e.g., an immmobilized glucoseisomerase (such as Sweetzyme™)

At least 3 enzymatic improvements of this process could be envisaged.All three improvements could be seen as individual benefits, but anycombination (e.g., 1+2, 1+3, 2+3 or 1+2+3) could be employed:

Improvement 1. Reduction of the Calcium Dependency of the LiquefyingAlpha-Amylase

Addition of free calcium is required to ensure adequately high stabilityof the alpha-amylase, but free calcium strongly inhibits the activity ofthe glucose isomerase and needs to be removed, by means of an expensiveunit operation, to an extent which reduces the level of free calcium tobelow 3-5 ppm. Cost savings could be obtained if such an operation couldbe avoided and the liquefaction process could be performed withoutaddition of free calcium ions.

To achieve that, a less calcium-dependent Termamyl-like alpha-amylasewhich is stable and highly active at low concentrations of free calcium(<40 ppm) is required. Such a Termamyl-like alpha-amylase should have apH optimum at a pH in the range of 4.5-6.5, preferably in the range of4.5-5.5.

Improvement 2. Reduction of Formation of Unwanted Maillard Products

The extent of formation of unwanted Maillard products during theliquefaction process is dependent on the pH. Low pH favours reducedformation of Maillard products. It would thus be desirable to be able tolower the process pH from around pH 6.0 to a value around pH 4.5;unfortunately, all commonly known, thermostable Termamyl-likealpha-amylases are not very stable at low pH (i.e., pH<6.0) and theirspecific activity is generally low.

Achievement of the above-mentioned goal requires a Termamyl-likealpha-amylase which is stable at low pH in the range of 4.5-5.5 and atfree calcium concentrations in the range of 0-40 ppm, and whichmaintains a high specific activity.

Improvement 3

It has been reported previously (U.S. Pat. No. 5,234,823) that whensaccharifying with A. niger glucoamylase and B. acidopullulyticuspullulanase, the presence of residual alpha-amylase activity from theliquefaction process can lead to lower yields of dextrose if thealpha-amylase is not inactivated before the saccharification stage. Thisinactivation can typically be carried out by adjusting the pH to below4.3 at 95° C., before lowering the temperature to 60° C. forsaccharification.

The reason for this negative effect on dextrose yield is not fullyunderstood, but it is assumed that the liquefying alpha-amylase (forexample Termamyl™ 120 L from B. licheniformis) generates “limitdextrins” (which are poor substrates for B. acidopullulyticuspullulanase) by hydrolyzing 1,4-alpha-glucosidic linkages close to andon both sides of the branching points in amylopectin. Hydrolysis ofthese limit dextrins by glucoamylase leads to a build-up of thetrisaccharide panose, which is only slowly hydrolyzed by glucoamylase.

The development of a thermostable alpha-amylase which does not sufferfrom this disadvantage would be a significant process improvement, as noseparate inactivation step would be required.

If a Termamyl-like, low-pH-stable alpha-amylase is developed, analteration of the specificity could be an advantage needed incombination with increased stability at low pH.

The methodology and principles of the present invention make it possibleto design and produce variants according to the invention havingrequired properties as outlined above. In this connection, particularlyinteresting mutations are mutations in a Termamyl-like alpha-amylase[for example Termamyl™ itself (B. licheniformis alpha-amylase; SEQ IDNO: 2); or a Termamyl-like alpha-amylase having an N-terminal amino acidsequence (i.e., the partial sequence up to the amino acid positioncorresponding to position 35 in Termamyl™) which is identical to that inB. amyloliquefaciens alpha-amylase (SEQ ID NO: 4), i.e., a Termamyl-likealpha-amylase having the following N-terminal sequence relative to aminoacid sequence of Termamyl™:

-   A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I, where an    asterisk (*) indicates deletion of the amino acid residue in    question] at positions corresponding to any of the following    positions in Termamyl™:-   H133-   H156-   A181-   A209-   G310-   H450-   V128-   N104-   V54-   S187-   H293-   A294    (where each of the latter amino acid residues may be replaced by any    other amino acid residue, i.e., any other residue chosen among A, R,    N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V), as well as    the following triple deletions:-   K370*+G371*+D372*-   D372*+S373*+Q374*

Particularly preferred substitutions at the above-indicated positionsare the following:

-   H133I-   H156Y-   A181T-   A209V-   G310D-   H450Y-   V128E-   N104D-   V54W,Y,F,I,L-   S187D-   H293Y-   A294V.

Any combination of one or more (i.e., one, two, three, four, etc.) ofthe above indicated mutations may appropriately be effected in aTermamyl-like alpha-amylase in the context in question, and particularlyinteresting variants of the invention in the context of achieving one ormore of the above-mentioned improvements in relation to the starchliquefaction behavior of alpha-amylases include variants comprisingcombinations of multiple mutations corresponding to the followingcombinations of mutations in Termamyl™ (SEQ ID NO: 2) itself:

-   V54W+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G371*+D372*+H450Y;-   V54W+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S373*+Q374*+H450Y+;-   V54Y+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*    +G371*+D372*+H450Y;-   V54Y+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S373*+Q374*+H450Y;-   V54F+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G371*+D372*+H450Y;-   V54F+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S373*+Q374*+H450Y;-   V54I+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G371*+D372*+H450Y;-   V54I+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S373*+Q374*+H450Y;-   V54L+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G371*+D372*+H450Y;

Further interesting variants of the invention in this context includevariants comprising single or multiple mutations corresponding to thefollowing single or multiple mutations in Termamyl™ itself:

-   mutations (amino acid substitutions) at positions N172 (e.g.,    N172R,K), S187 (e.g., S187D), N188 (e.g., N188P), N190 (e.g.,    N190L,F), H205 (e.g., H205C), D207 (e.g., D207Y), A210 (e.g.,    A210S), Q264 (e.g., Q264S) or N265 (e.g., N265Y);-   the following multiple mutations:-   H156Y+A181T+A209V;-   H156Y+A181T+N190F+A209V+Q264S;-   A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A209V;-   A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N190F+A209V;    or-   A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N190F+A209V+Q264S    as well as combinations of any two or more of the latter single or    multiple mutations.

As already indicated, numerous variants according to the invention areparticularly well suited for use in starch conversion, e.g., in starchliquefaction. In this connection, a further aspect of the presentinvention relates to compositions comprising a mixture of:

(i) the alpha-amylase from B. licheniformis having the sequence shown inSEQ ID NO: 2 with one or more variants (mutant alpha-amylases) accordingto the invention derived from (as the parent Termamyl-likealpha-amylase) the B. stearothermophilus alpha-amylase having thesequence shown in SEQ ID NO: 6; or

(ii) the alpha-amylase from B. stearothermophilus having the sequenceshown in SEQ ID NO: 6 with one or more variants (mutant alpha-amylases)according to the invention derived from one or more other parentTermamyl-like alpha-amylases (e.g., from the B. licheniformisalpha-amylase having the sequence shown in SEQ ID NO: 2, or from one ofthe other parent Termamyl-like alpha-amylases specifically referred toherein); or

(iii) one or more variants (mutant alpha-amylases) according to theinvention derived from (as the parent Termamyl-like alpha-amylase) theB. stearothermophilus alpha-amylase having the sequence shown in SEQ IDNO: 6 with one or more variants (mutant alpha-amylases) according to theinvention derived from one or more other parent Termamyl-likealpha-amylases (e.g., from the B. licheniformis alpha-amylase having thesequence shown in SEQ ID NO: 2, or from one of the other parentTermamyl-like alpha-amylases specifically referred to herein).

Preferred mutations in a variant of B. stearothermophilus alpha-amylaseto be incorporated in such a mixture include substitutions at N193and/or at E210, and/or the pairwise deletions R179*+G180* or I181*+G182*(using the numbering of the amino acid sequence for this particularalpha-amylase).

Compositions of one of the latter types, containing B.stearothermophilus alpha-amylase or a variant thereof according to theinvention, appear to have great potential for use in starchliquefaction. The ratio (expressed, e.g., in terms of mg of activeamylolytic protein per liter of liquid medium) between the individualalpha-amylolytic components of a given mixture will depend on the exactnature and properties of each component.

Detergent Compositions

As mentioned above, variants of the invention may suitably beincorporated in detergent compositions. Reference is made, for example,to WO 96/23874 and WO 97/07202 for further details concerning relevantingredients of detergent compositions (such as laundry or dishwashingdetergents), appropriate methods of formulating the variants in suchdetergent compositions, and for examples of relevant types of detergentcompositions.

Detergent compositions comprising a variant of the invention mayadditionally comprise one or more other enzymes, such as a lipase,cutinase, protease, cellulase, peroxidase or laccase, and/or anotheralpha-amylase.

Alpha-amylase variants of the invention may be incorporated indetergents at conventionally employed concentrations. It is at presentcontemplated that a variant of the invention may be incorporated in anamount corresponding to 0.00001-1 mg (calculated as pure, active enzymeprotein) of alpha-amylase per liter of wash/dishwash liquor usingconventional dosing levels of detergent.

Materials and Methods

Construction of pSNK101

This E. coli/Bacillus shuttle vector can be used to introduce mutationswithout expression of alpha-amylase in E. coli and then be modified insuch way that the alpha-amylase is active in Bacillus. The vector wasconstructed as follows: The alpha-amylase gene in the pX vector (pDN1528with the following alterations within amyL: BAN(1-33), H156Y, A181T,N190F, A209V, Q264S, the plasmid pDN1528 is further described inExample 1) was inactivated by interruption in the Pstl site in the 5′coding region of the alpha-amylase gene by a 1.2 kb fragment containingan E. coli origin fragment. This fragment was amplified from the pUC19(GenBank Accession #: X02514) using the forward primer:5′-gacctgcagtcaggcaacta-3′ (SEQ ID NO: 8) and the reverse primer:5′-tagagtcgacctgcaggcat-3′ (SEQ ID NO: 9). The PCR amplicon and the pXplasmid containing the alpha-amylase gene were digested with Pstl at 37°C. for 2 hrs. The pX vector fragment and the E. coli origin ampliconwere ligated at room temperature for 1 hour and transformed in E. coliby electrotransformation. The resulting vector is designated pSnK101.

Fermentation and Purification of Alpha-amylase Variants

A B. subtilis strain harbouring the relevant expression plasmid isstreaked on an LB-agar plate with 15 micrograms/mI chloramphenicol from−80° C. stock, and grown overnight at 37° C.

The colonies are transferred to 100 ml BPX media supplemented with 15micrograms/ml chloramphenicol in a 500 ml shaking flask.

Composition of BPX medium:

Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/l Sodiumcaseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12 H₂O 9 g/l Pluronic ™0.1 g/l

The culture is shaken at 37° C. at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on a UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM acetate pH 5.5. The UF-filtrate is applied onan S-sepharose F.F. and elution is carried out by step elution with 0.2M NaCl in the same buffer. The eluate is dialyzed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3 M NaCl over 6 column volumes. The fractions which contain theactivity (measured by the Phadebas assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by a treatment with 0.5% w/vol.active coal in 5 minutes.

Assay for Alpha-amylase Activity

Alpha-amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-colouredstarch polymer which has been mixed with bovine serum albumin and abuffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

General Method for Random Mutagenesis by Use of the DOPE Program

The random mutagenesis may be carried out by the following steps:

-   1. Select regions of interest for modification in the parent enzyme-   2. Decide on mutation sites and nonmutated sites in the selected    region-   3. Decide on which kind of mutations should be carried out, e.g.,    with respect to the desired stability and/or performance of the    variant to be constructed-   4. Select structurally reasonable mutations.-   5. Adjust the residues selected by step 3 with regard to step 4.-   6. Analyze by use of a suitable dope algoritm the nucleotide    distribution.-   7. If necessary, adjust the wanted residues to genetic code realism    (e.g., taking into account constraints resulting from the genetic    code (e.g., in order to avoid introduction of stop codons)) (the    skilled person will be aware that some codon combinations cannot be    used in practice and will need to be adapted)-   8. Make primers-   9. Perform random mutagenesis by use of the primers-   10. Select resulting alpha-amylase variants by screening for the    desired improved properties.

Suitable dope algorithms for use in step 6 are well known in the art.One algorithm is described by Tomandl et al., 1997, Journal ofComputer-Aided Molecular Design 11: 29-38. Another algorithm, DOPE, isdescribed in the following:

The Dope Program

The “DOPE” program is a computer algorithm useful to optimize thenucleotide composition of a codon triplet in such a way that it encodesan amino acid distribution which resembles most the wanted amino aciddistribution. In order to assess which of the possible distributions isthe most similar to the wanted amino acid distribution, a scoringfunction is needed. In the “Dope” program the following function wasfound to be suited:

${s \equiv {\prod\limits_{i = 1}^{N}\;\left( {\frac{x_{i}^{y_{i}}}{y_{i}^{y_{i}}}\frac{\left( {1 - x_{i}} \right)^{1 - y_{i}}}{\left( {1 - y_{i}} \right)^{1 - y_{i}}}} \right)^{w_{i}}}},$

where the x_(i)'s are the obtained amounts of amino acids and groups ofamino acids as calculated by the program, y_(i)'s are the wanted amountsof amino acids and groups of amino acids as defined by the user of theprogram (e.g., specify which of the 20 amino acids or stop codons arewanted to be introduced, e.g., with a certain percentage (e.g., 90% Ala,3% Ile, 7% Val), and w_(i)'s are assigned weight factors as defined bythe user of the program (e.g., depending on the importance of having aspecific amino acid residue inserted into the position in question). Nis 21 plus the number of amino acid groups as defined by the user of theprogram. For purposes of this function 0⁰ is defined as being 1.

A Monte-Carlo algorithm (one example being the one described by Valleau,J. P. & Whittington, S. G. (1977) A guide to Mont Carlo for statisticalmechanics: 1 Highways. In “Stastistical Mechanics, Part A” EqulibriumTechniqeues ed. B. J. Berne, New York: Plenum) is used for finding themaximum value of this function. In each iteration the following stepsare performed:

-   1. A new random nucleotide composition is chosen for each base,    where the absolute difference between the current and the new    composition is smaller than or equal to d for each of the four    nucleotides G, A, T, C in all three positions of the codon (see    below for definition of d).-   2. The scores of the new composition and the current composition are    compared by the use of the function s as described above. If the new    score is higher or equal to the score of the current composition,    the new composition is kept and the current composition is changed    to the new one. If the new score is smaller, the probability of    keeping the new composition is exp(1000(new_score−current_score)).

A cycle normally consists of 1000 iterations as described above in whichd is decreasing linearly from 1 to 0. One hundred or more cycles areperformed in an optimization process. The nucleotide compositionresulting in the highest score is finally presented.

EXAMPLE 1

Construction of Termamyl™ Variants in Accordance with the Invention

Termamyl (B. licheniformis alpha-amylase SEQ ID NO: 2) is expressed inB. subtilis from a plasmid denoted pDN1528. This plasmid contains thecomplete gene encoding Termamyl, amyL, the expression of which isdirected by its own promoter. Further, the plasmid contains the originof replication, ori, from plasmid pUB110 and the cat gene from plasmidpC194 conferring resistance towards chloramphenicol. pDN1528 is shown inFIG. 9 of WO 96/23874.

A specific mutagenesis vector containing a major part of the codingregion of SEQ ID NO: 1 was prepared. The important features of thisvector, denoted pJeEN1, include an origin of replication derived fromthe pUC plasmids, the cat gene conferring resistance towardschloramphenicol, and a frameshift-containing version of the bla gene,the wild type of which normally confers resistance towards ampicillin(amp^(R) phenotype). This mutated version results in an amp^(S)phenotype. The plasmid pJeEN1 is shown in FIG. 10 of WO 96/23874, andthe E. coli origin of replication, ori, bla, cat, the 5′-truncatedversion of the Termamyl amylase gene, and selected restriction sites areindicated on the plasmid.

Mutations are introduced in amyL by the method described by Deng andNickoloff (1992, Anal. Biochem. 200: 81-88) except that plasmids withthe “selection primer” (primer #6616; see below) incorporated areselected based on the amp^(R) phenotype of transformed E. coli cellsharboring a plasmid with a repaired bla gene, instead of employing theselection by restriction enzyme digestion outlined by Deng andNickoloff. Chemicals and enzymes used for the mutagenesis were obtainedfrom the ChameleonÔ mutagenesis kit from Stratagene (catalogue number200509).

After verification of the DNA sequence in variant plasmids, thetruncated gene, containing the desired alteration, is subcloned intopDN1528 as a Pstl-EcoRl fragment and transformed into the protease- andamylase-depleted Bacillus subtilis strain SHA273 (described in WO92/11357 and WO 95/10603) in order to express the variant enzyme.

The Termamyl variant V54W was constructed by the use of the followingmutagenesis primer (written 5′ to 3′, left to right):

(SEQ ID NO: 10) PG GTC GTA GGC ACC GTA GCC CCA ATC CGC TTG

The Termamyl variant A52W+V54W was constructed by the use of thefollowing mutagenesis primers (written 5′ to 3′, left to right):

(SEQ ID NO: 11) PG GTC GTA GGC ACC GTA GCC CCA ATC CCA TTG GCT CG

Primer #6616 (written 5′ to 3′, left to right; P denotes a 5′phosphate):

(SEQ ID NO: 12) P CTG TGA CTG GTG AGT ACT CAA CCA AGT C

The Termamyl variant V54E was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 13) PGG TCG TAG GCA CCG TAG CCC TCA TCC GCT TG

The Termamyl variant V54M was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 14) PGG TCG TAG GCA CCG TAG CCC ATA TCC GCT TG

The Termamyl variant V54I was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 15) PGG TCG TAG GCA CCG TAG CCA ATA TCC GCT TG

The Termamyl variants Y290E and Y290K were constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 16) PGC AGC ATG GAA CTG CTY ATG AAG AGG CAC GTC AAA CY represents an equal mixture of C and T. The presence of a codonencoding either glutamate or lysine in position 290 was verified by DNAsequencing.

The Termamyl variant N190F was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 17) PCA TAG TTG CCG AAT TCA TTG GAA ACT TCC C

The Termamyl variant N188P+N190F was constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 18) PCA TAG TTG CCG AAT TCA GGG GAA ACT TCC CAA TC

The Termamyl variant H140K+H142D was constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 19) PCC GCG CCC CGG GAA ATC AAA TTT TGT CCA GGC TTT AAT TAG

The Termamyl variant H156Y was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 20) PCA AAA TGG TAC CAA TAC CAC TTA AAA TCG CTG

The Termamyl variant A181T was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 21) PCT TCC CAA TCC CAA GTC TTC CCT TGA AAC

The Termamyl variant A209V was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 22) PCTT AAT TTC TGC TAC GAC GTC AGG ATG GTC ATA ATC

The Termamyl variant Q264S was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 23) PCG CCC AAG TCA TTC GAC CAG TAC TCA GCT ACC GTA AAC

The Termamyl variant S187D was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 24) PGC CGT TTT CAT TGT CGA CTT CCC AAT CCC

The Termamyl variant DELTA(K370-G371-D372) (i.e., deleted of amino acidresidues nos. 370, 371 and 372) was constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 25) PGG AAT TTC GCG CTG ACT AGT CCC GTA CAT ATC CCC

The Termamyl variant DELTA(D372-S373-Q374) was constructed by the use ofthe following mutagenesis primer (written 5′-3′, left to right):

(SEQ ID NO: 26) PGG CAG GAA TTT CGC GAC CTT TCG TCC CGT ACA TAT C

The Termamyl variants A181T and A209V were combined to A181T+A209V bydigesting the A181T containing pDN1528-like plasmid (i.e., pDN1528containing within amyL the mutation resulting in the A181T alteration)and the A209V-containing pDN1528-like plasmid (i.e., pDN1528 containingwithin amyL the mutation resulting in the A209V alteration) withrestriction enzyme ClaI which cuts the pDN1528-like plasmids twiceresulting in a fragment of 1116 by and the vector-part (i.e., containsthe plasmid origin of replication) of 3850 bp. The fragment containingthe A209V mutation and the vector part containing the A181T mutationwere purified by QIAquick gel extraction kit (purchased from QIAGEN)after separation on an agarose gel. The fragment and the vector wereligated and transformed into the protease and amylase depleted Bacillussubtilis strain referred to above. Plasmid from amy+ (clearing zones onstarch containing agar-plates) and chloramphenicol resistanttransformants were analysed for the presence of both mutations on theplasmid.

In a similar way as described above, H156Y and A209V were combinedutilizing restriction endonucleases Acc65I and EcoRI, givingH156Y+A209V.

H156Y+A209V and A181T+A209V were combined into H156Y+A181T+A209V by theuse of restriction endonucleases Acc65I and HindIII.

The 35 N-terminal residues of the mature part of Termamyl variantH156Y+A181T+A209V were substituted by the 33 N-terminal residues of theB. amyloliquefaciens alpha-amylase (SEQ ID NO: 4) (which in the presentcontext is termed BAN) by a SOE-PCR approach (Higuchi et al., 1988,Nucleic Acids Research 16: 7351) as follows:

Primer 19364 (sequence 5′-3′): (SEQ ID NO: 27)CCT CAT TCT GCA GCA GCA GCC GTA AAT GGC ACG CTG Primer 19362:(SEQ ID NO: 28) CCA GAC GGC AGT AAT ACC GAT ATC CGA TAA ATG TTC CGPrimer 19363: (SEQ ID NO: 29) CGG ATA TCG GTA TTA CTG CCG TCT GGA TTCPrimer 1C: (SEQ ID NO: 30) CTC GTC CCA ATC GGT TCC GTC

A standard PCR, polymerase chain reaction, was carried out using the Pwothermostable polymerase from Boehringer Mannheim according to themanufacturer's instructions and the temperature cycles: 5 minutes at 94°C., 25 cycles of (94° C. for 30 seconds, 50° C. for 45 seconds, 72° C.for 1 minute), 72° C. for 10 minutes.

An approximately 130 by fragment was amplified in a first PCR denotedPCR1 with primers 19364 and 19362 on a DNA fragment containing the geneencoding the B. amyloliquefaciens alpha-amylase.

An approximately 400 by fragment was amplified in another PCR denotedPCR2 with primers 19363 and 1C on template pDN1528.

PCR1 and PCR2 were purified from an agarose gel and used as templates inPCR3 with primers 19364 and 1C, which resulted in a fragment ofapproximately 520 bp. This fragment thus contains one part of DNAencoding the N-terminus from BAN fused to a part of DNA encodingTermamyl from the 35th amino acid.

The 520 by fragment was subcloned into a pDN1528-like plasmid(containing the gene encoding Termamyl variant H156Y+A181T+A209V) bydigestion with restriction endonucleases PstI and SadII, ligation andtransformation of the B. subtilis strain as previously described. TheDNA sequence between restriction sites PstI and SacII was verified byDNA sequencing in extracted plasmids from amy+ and chloramphenicolresistant transformants.

The final construct containing the correct N-terminus from BAN andH156Y+A181T+A209V was denoted BAN(1-35)+H156Y+A181T+A209V.

N190F was combined with BAN (1-35)+H156Y+A181T+A209V givingBAN(1-35)+H156Y+A181T+N190F+A209V by carrying out mutagenesis asdescribed above except that the sequence of amyL in pJeEN1 wassubstituted by the DNA sequence encoding Termamyl variantBAN(1-35)+H156Y+A181T+A209V.

Q264S was combined with BAN (1-35)+H156Y+A181T+A209V givingBAN(1-35)+H156Y+A181T+A209V+Q264S by carrying out mutagenesis asdescribed above except that the sequence of amyL in pJeEN wassubstituted by the DNA sequence encoding Termamyl variantBAN(1-35)+H156Y+A181T+A209V.

BAN(1-35)+H156Y+A181T+A209V+Q264S and BAN(1-35)+H156Y+A181T+N190F+A209Vwere combined into BAN(1-35)+H156Y+A181T+N190F+A209V+Q264S utilizingrestriction endonucleases BsaHI (BsaHI site was introduced close to theA209V mutation) and PstI.

EXAMPLE 2

Construction, by Localized Random, Doped Mutagenesis, of Termamyl-likeAlpha-amylase Variants Having an Improved Stability at Low Ph and aReduced Dependency on Calcium Ions for Stability Compared to the ParentEnzyme

Alpha-amylases are of great importance for the industrial starchliquefaction process. The variant of the thermostable B. licheniformisalpha-amylase consisting of amino acids 1-33 of the B. amyloliquefaciensamylase (SEQ ID NO: 4) fused to amino acids 36-483 of the B.licheniformis amylase (SEQ ID NO: 2) and further comprising thefollowing mutations: Y156, T181, F190, V209 and S264 (the constructionof this variant is described in Example 1) has a very satisfactorystability at low pH and low calcium concentrations. In an attempt tofurther improve the stability at low pH and low calcium concentration ofsaid alpha-amylase variant random mutagenesis in preselected regions wasperformed.

The regions were:

Region: Residue: I: Phe153-Thr163 II: Gln178-Asn192 III: His205-Arg214IV: Ala232-Asp237 and VIII: Gly131-Lys136 IX: Asp164-Tyr175 X: Tyr262-Thr278 Region Total % Mean % Number of residues changed I: 35 88 8out of 11 II: 20 86 11 out of 15 III: 27 88 10 out of 10 IV: 34 91 11out of 12 VIII: 39 86 6 out of 6 IX: 46 93 12 out of 12 X: 27 90 12 outof 13 VIII + IX: 18 VIII + IX + II: 4 II + III + IV: 2 IV + I: 12

The numbers under Total % give the total number of wild-type (wt) aminoacids desired in a given region after doping. The number is obtained bymultiplication of the number of mutated positions (e.g., 8 with respectto region I) by their respective wt. With respect to region I thedesired total % is 80*80*90*90*90*90*95*90/100=35%.

The Mean % is the mean doping level for the total number of positions ofthe region in question (e.g., 11 positions with respect to region I).For region I the mean % is calculated as follows:80+80+90+90+90+90+95+90=705 divided by 11=88%

The DOPE software (see Materials and Methods) was used to determinespiked codons for each suggested change in the seven regions minimizingthe amount of stop codons. The exact distribution of nucleotides wascalculated in the three positions of the codon to give the suggestedpopulation of amino acid changes. The doped regions were dopedspecifically in the indicated positions to have a high chance of gettingthe desired residues, but still allow other possibilities.

For instance, the original H156 in the wt sequence was mutated into a Y,meaning a new codon, and then doped 10% for other residues. That is theDNA sequence has the code for a Y instead for a H. In position 156 theTyr has been programmed to be 90% desired and other residues has beenfreely allowed. For some positions it was not possible to create thesuggested population of amino acid residues because the genetic coderestricted the structurally and functionally desired residues. Theresulting seven doped oligonucleotides are shown in tables 1-7: with thewt nucleotide and amino acid sequences and the distribution ofnucleotides for each doped position. All library primers weresynthesized as sense strands.

TABLE 1 Library DASI (Phe153-Thr163)153 154 155 156 157 158 159 160 161 162 163Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly ThrPrimer: 5′-CGC GGC AGC ACA TAC AGC GAT T1T 2A3 TGG45T TGG 67T 8AT TTT GAC GGA A9C GAT TGG GAC GAG TCC CGA AAG-3′(SEQ ID NO: 31)

Distribution of nucleotides for each doped position.

-   1: 80% T, 20% A.-   2: 96% A, 2% G, 2% C.-   3: 98% A, 2% T.-   4: 93% T, 4% G, 3% A.-   5: 97% A, 3% G.-   6: 98% T, 2% A.-   7: 97% A, 3% C.-   8: 90% C, 10% T.-   9: 95% C, 5% A.

TABLE 2 Library DASII (Gln178-Asn192)178 179 180 181 182 183 184 185 186 187 188 189Gln Gly Lys Thr Trp Asp Trp Glu Val Ser Asn Glu 190 191 192 Phe Gly AsnPrimer: 5′-CTG AAC CGC ATC TAT AAG TTT 1A2 34T AAG567 TGG GAT 89G GA10 GTT A11T 1213T GAA T1415161718 AAC TAT GAT TAT TTG ATG TAT-3′ (SEQ ID NO: 32)

Distribution of nucleotides for each doped position.

-   1: 93% C, 7% A.-   2: 84% G, 16% T.-   3: 95% G, 5% A.-   4: 95% G, 5% C.-   5: 94% A, 6% G.-   6: 95% C, 5% A.-   7: 62% T, 38% G.-   8: 87% T, 13% A.-   9: 91% G, 9% C.-   10: 92% G, 8% T.-   11: 90% G, 5% A, 5% C.-   12: 88% A, 12% C.-   13: 88% A, 12% C.-   14: 93% T, 5% A, 2% C.-   15: 97% T, 3% G.-   16: 86% G, 14% A.-   17: 89% G, 11% C.-   18: 60% G, 40% T.

TABLE 3 Library DASIII (His205-Arg214)205 206 207 208 209 210 211 212 213 214His Pro Asp Val Val Ala Glu Ile Lys ArgPrimer: 5′-TAT GCC GAC ATC GAT TAT GAC 12T 3CT 4567TT 8910 1112T 13A14 15T16 A17A 1819A TGG GGC ACT TGG TAT GCC AAT-3′(SEQ ID NO: 33)

Distribution of nucleotides for each doped position.

-   1: 89% C, 11% T.-   2: 89% A, 11% G.-   3: 95% C, 2.5% T, 2.5% A.-   4: 96% G, 1% A, 3% T.-   5: 96% A, 4% C.-   6: 98% T, 2% A.-   7: 95% G, 2.5% A, 2.5% C.-   8: 93% G, 7% A.-   9: 96% T, 4% A.-   10: 84% A, 16% G.-   11: 81% G, 7% A, 7% T, 5% C.-   12: 98% C, 2% A.-   13: 96% G, 4% C.-   14: 94% G, 6% T.-   15: 82% A, 18% T.-   16: 50% A, 50% T.-   17: 90% A, 10% G.-   18: 70% A, 30% C.-   19: 86% G, 14% A.

TABLE 4 Library DASIV (Ala232-Asp243)232 233 234 235 236 237 238 239 240 241 242 243Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg AspPrimer: 5′-TTG GAC GGT TTC CGT CTT GAT 12T G3T AAA456 7TT A8G T9T 1011T T12T 13T14 1516G GA17 TGGGTT AAT CAT GTC AGG GAA-3′ (SEQ ID NO: 34)

Distribution of nucleotides for each doped position.

-   1: 93% G, 3.5% A, 3.5% T.-   2: 94% C, 4% T.-   3: 94% T, 6% C.-   4: 93% C, 2% T, 2% A, 3% G.-   5: 98% A, 2% T.-   6: 98% T, 2% A.-   7: 95% A, 5% C.-   8: 94% A, 6% G.-   9: 90% T, 10% A.-   10: 89% T, 11% A.-   11: 89% C, 11% A.-   12: 95% T, 5% A-   13: 64% C, 33% T, 3% A.-   14: 93% A, 7% T.-   15: 90% A, 10% C.-   16: 90% G, 5% A, 5% C-   17: 90% T, 10% A

TABLE 5 Library DASVIII (Gly131-Lys136) 131 132 133 134 135 136Gly Glu His Leu Ile LysPrimer: 5′-GCT GAC CGC AAC CGC GTA ATT TCA 123 GA456T 78A 9TA A10G GCC TGG ACA CAT TTT CAT TTT-3′ (SEQ ID NO: 35)

Distribution of nucleotides for each doped position.

-   1: 91% G, 9% A.-   2: 87% G, 13% C.-   3: 90% T, 10% G.-   4: 90% G, 10% T.-   5: 85% C, 8% T, 7% A.-   6: 89% A, 9% T, 2% C.-   7: 88% T, 12% A-   8: 88% T, 11% C, 1% G-   9: 92% A, 8% T-   10: 93% A, 7% G

TABLE 6 Library DASIX (Asp164-Tyr175)164 165 166 167 168 169 170 171 172 173 174 175Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile TyrPrimer: 5′-TGG TAC CAT TTT GAC GGA ACC GAT TGG 1A2GAG 3CG CGA A4G 56A A7T A8G 9 1011 T12T AAG TTT CAA GGA AAG GCT TGG-3′(SEQ ID NO: 36)

Distribution of nucleotides for each doped position.

-   1: 94% G, 6% A.-   2: 96% T, 4% G.-   3: 92% T, 4% A, 4% G.-   4: 95% A, 5% G.-   5: 93% C, 7% A.-   6: 92% T, 8% A.-   7: 90% A, 5% G,5% C.-   8: 90% G, 10% A.-   9: 92% A, 6% G, 2% T.-   10: 92% T, 8% A.-   11: 50% T, 50% C.-   12: 96% A, 4% T.

TABLE 7 Library DASX (Tyr262-Asn278)262 263 264 265 266 267 268 269 270 271 272 273Tyr Trp Ser Asn Asp Leu Gly Ala Leu Glu Asn Tyr 374 275 276 277 278Leu Asn Lys Thr Asn Primer: 5′-GAA ATG TTT ACG GTA GCT GAA T1T TGG 23456T 7A8 91011 1213T 1415T 16T17 GA18 A19T T20T21T22 A23C A24G ACA 25AT TTT AAT CAT TCA GTG TTT GAC-3′ (SEQ ID NO: 37)

Distribution of nucleotides for each doped position.

-   1: 95% A, 5% T.-   2: 97% A, 3% G.-   3: 95% G, 2.5% A, 2.5% C.-   4: 94% T, 6.2% G.-   5: 97% A, 3% T.-   6: 94% A, 3% G, 3% C.-   7: 95% G, 5% A.-   8: 95% T, 5% A.-   9: 52% T, 45% C, 3% A.-   10: 96% T, 4% C.-   11: 60% A, 40% G.-   12: 90% G, 10% A.-   13: 94% G, 6% C.-   14: 81% G, 8% A, 8% T, 3% C.-   15: 98% C, 2% T.-   16: 90% C, 10% A.-   17: 50% G, 50% T.-   18: 90% A, 10% T.-   19: 90% A, 5% G, 5% C.-   20: 95% A, 5% T.-   21: 91% T, 9% A.-   22: 92% A, 8% G.-   23: 94% A, 3% G, 3% C.-   24: 93% G, 7% A.-   25: 90% A, 10% G.    Random Mutagenesis

The spiked oligonucleotides apparent from Tables 1-7 (which by a commonterm is designated FDAS in FIG. 3) and reverse primers RDAS for eachregion and specific B. licheniformis primers covering the SacII and theSaII sites are used to generate PCR-library-fragments by the overlapextension method (Horton et al., 1989, Gene 77: 61-68) with an overlapof 21 bp. FIG. 3 shows the PCR strategy. The PCR fragments are cloned inthe E. coli/Bacillus shuttle vector pSNK101 (see Materials and Methods)enabling mutagenesis in E. coli and immediate expression in Bacillussubtilis preventing lethal accumulation of amylases in E. coli. Afterestablishing the cloned PCR fragments in E. coli, a modified pUC19fragment is digested out of the plasmid and the promoter and the mutatedTermamyl gene is physically connected and expression can take place inBacillus.

Screening

The seven libraries may be screened in the low pH and the low calciumfilter assays described below.

Low pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith 10 micrograms/ml chloramphenicol at 37° C. for at least 21 hrs. Thecellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter and the nitrocellulose filter with bound variants istransferred to a container with citrate buffer, pH 4.5 and incubated at80° C. for 15 minutes. The cellulose acetate filters with colonies arestored on the TY-plates at room temperature until use. After incubation,residual activity is detected on plates containing 1% agarose, 0.2%starch in citrate buffer, pH 6.0. The assay plates with nitrocellulosefilters are marked the same way as the filter sandwich and incubated for2 hours at 50° C. After removal of the filters the assay plates arestained with 10% Lugol solution. Starch degrading variants are detectedas white spots on dark blue background and then identified on thestorage plates. Positive variants are rescreened twice under the sameconditions as the first screen.

Low Calcium Filter Assay

The assay is performed in the same way as the low pH filter assay withthe following modifications: The filter with bound proteins is incubatedat 95° C., pH 6.0 for 1 hour. with different EDTA concentrations (0.001mM-100 mM).

The following variants were obtained by the above method (BAN designatesB. amyloliquefaciens alpha-amylase):

-   -   BAN/Termamyl hybrid*+H156Y+A181T+N190F+A209V+Q264S+E211Q    -   BAN/Termamyl        hybrid*+H156Y+A181T+N190F+A209V+Q264S+H205C+D207Y+A210S

The mutations indicated in bold were introduced by the randommutagenesis method. The stability data for these variants appear fromTable 11 in Example 3.

In an analogous manner to that described above, random mutagenesis ofthe above identified seven regions are performed on the parent B.licheniformis alpha-amylase (SEQ ID NO: 2). The doping scheme isdetermined analogously to that used above.

EXAMPLE 3

Measurement of the Calcium- and pH-Dependent Stability

Normally, the industrial liquefaction process runs using pH 6.0-6.2 asliquefaction pH and an addition of 40 ppm free calcium in order toimprove the stability at 95-105° C. Some of the herein proposedsubstitutions have been made in order to improve the stability at

-   1. lower pH than pH 6.2 and/or-   2. at free calcium levels lower than 40 ppm free calcium.

Three different methods have been used to measure the improvements instability obtained by the different substitutions in Termamyl:

-   1. One assay which measures the stability at slightly reduced pH, pH    5.5, in the presence of 40 ppm free calcium. (thereby, the    improvement of stability at low pH is measured). 10 micrograms of    the variant were incubated under the following conditions: A 0.1 M    acetate solution, pH adjusted to pH 5.5, containing 40 ppm calcium    and 5% w/w common corn starch (free of calcium). Incubation was made    in a water bath at 95° C. for 30 minutes.-   2. Another assay which measures the stability in the absence of free    calcium and where the pH is maintained at pH 6.2. This assay    measures the decrease in calcium sensitivity: 10 micrograms of the    variant were incubated under the following conditions: A 0.1 M    acetate solution, pH adjusted to pH 6.2, containing 5% w/w common    corn starch (free of calcium). Incubation was made in a water bath    at 95° C. for 30 minutes.-   3. A third assay wherein the conditions of assays nos. 1 and 2 have    been combined. This assay measures the stability in the absence of    calcium and at low pH (pH 5.5).-   4. A fourth assay similar to no. 3. where the pH has been further    reduced to pH 5.0.    Stability Determination

All the stability trials 1, 2, 3 and 4 have been made using the same setup. The method was:

The enzyme was incubated under the relevant conditions (1-4). Sampleswere taken at 0, 5, 10, 15 and 30 minutes and diluted 25 times (samedilution for all taken samples) in assay buffer (0.1 M 50 mM Brittonbuffer pH 7.3) and the activity was measured using the Phadebas assay(Pharmacia) under standard conditions pH 7.3, 37° C.

The activity measured before incubation (0 minutes) was used asreference (100%). The decline in percent was calculated as a function ofthe incubation time. The table shows the residual activity after 30minutes of incubation.

Specific Activity Determination

The specific activity was determined using the Phadebas assay(Pharmacia) as activity/mg enzyme. The activity was determined using thealpha-amylase assay described in the Materials and Methods section.

Results:

TABLE 8 Stability method no. 1./Low pH stability improvement ResidualActivity Specific activity, 37° after 30 minutes C., pH 7.3. PercentVariant of incubation relative to Termamyl Termamyl wt.  5% 100% H156Y15% 100% A209V 18% 100% Q264S 30% 130% H156Y + A181T + A209V 39% 100%H133Y + H156Y + A181T + 58%  85% A209V + H293Y + A294V + G310D + H450YBAN/Termamyl hybrid* + 48% 115% H156Y + A181T + A209V

TABLE 9 Stability method no. 2./decreased calcium sensitivity residualactivity Specific activity, 37° after 30 minutes C., pH 7.3. PercentVariant of incubation relative to Termamyl Termamyl wt. 52% — H156Y 70%— A209V 75% — A181T 57% 115% N190F 34% 140% N190F + N188P 57% 190%

TABLE 10 Stability method no. 3. Low pH stability improvement +decreased calcium sensitivity Residual Activity Specific activity, after30 minutes 37° C., pH 7.3 Variant of incubation Units/mg enzyme Termamylwt.  3% — BAN/Termamyl hybrid * + 20% — H156Y + A181T + A209V Q264S  5%— H140K + H142D  5% 115% V128E   50% ** 115% BAN/Termamyl hybrid * + 39%170% H156Y + A181T + N190F + A209V BAN/Termamyl hybrid * + 29% 175%H156Y + A181T + A209V + Q264S BAN/Termamyl hybrid * + 57% 210% H156Y +A181T + N190F + A209V + Q264S * BAN (B. amyloliquefaciens alpha-amylase(SEQ ID NO: 4)/Termamyl (B. licheniformis alpha-amylase (SEQ ID NO: 2)PCR hybrid. First 33 N-terminal amino acids are BAN and the rest 36-483are Termamyl (the construction of the variant is described in Example1). ** Measured after 5 minutes of incubation. Compared to Termamyl wt.which under same conditions shows 36% residual activity.

TABLE 11 Stability method no. 4./Low pH stability improvement (pH 5.0) +decreased calcium sensitivity Residual Activity Specific activity, after30 minutes 37° C., pH 7.3 Variant of incubation Units/mg enzymeBAN/Termamyl hybrid * +  9% 210% H156Y + A181T + N190F + A209V + Q264SBAN/Termamyl hybrid * + 28% 160% H156Y + A181T + N190F + A209V + Q264S +E211Q BAN/Termamyl hybrid * + 33% 130% H156Y + A181T + N190F + A209V +Q264S + H205C + D207Y + A210S * as indicated in relation to Table 10

The variants in the above Table 11 were constructed by means of thelocalized random mutagenesis described in Example 2.

EXAMPLE 4

Alpha-amylase Stability at Low pH and High Temperature

This example summarises the stability results of variants characterisedby a fluorimetric assay at 70° C. under two different conditions, (1) pH4.5 and 1 mM CaCl₂ and (2) pH 6.2 and 10 micro-M CaCl₂.

Description of Method

All fluorescence experiments were performed on a Perkin-Elmer LS-50luminescence spectrometer using a 4-cuvette holder. The temperature wascontrolled by a circulating water-bath and measured directly in thecuvette using a Noronix Digital Thermometer (model NTD 100). Duringmeasurements, thorough mixing of reagents in the cuvette was ensuredusing magnetic stirrers operating at high stirring rate. The cuvetteswere capped with teflon-lids to minimize evaporation.

Intrinsic protein-fluorescence (due to Trp side-chains) was monitored byexcitation at 280 nm and emission at 350 nm. Slit-widths were 5 nm.

During kinetic measurements, 4 reactions were monitored in parallel.Data was collected in the Wavelength Programme dialogue, allowingautomatic data-collection over a prolonged period (e.g., over an hour).

Unfolding was carried out at 70° C. Unfolding conditions were

-   (1) 50 mM NaOAc pH 4.5 and 1 mM CaCl₂-   (2) 50 mM NaOAc pH 6.2 and 10 micro-M CaCl₂.    Protein concentration was 5 micrograms/ml and glycerol was at 0.5%    w/v (from protein stock solution).

Note: There was some variation from day to day in the absolute value ofthe unfolding half times due to slight temperature variations(occasioned by, e.g., different amounts of water in the water bath).However, Termamyl was always included as one of the four enzymesanalyzed in each experiment, in effect making it an internal standard.Unfolding rates relative to this internal standard were satisfactorilyreproducible (performed in triplicate).

Data analysis was carried out using GraphPad Prism software.

At pH 4.5, unfolding data could be fitted very satisfactorily to asingle-exponential decay with drift:F(t)=A*exp(−ln(2)*t/t _(1/2))+drift*t+offset  (1)where F is the measured fluorescence, A is the amplitude of theunfolding, t is time and t_(1/2) is the half-time of unfolding.

At pH 6.2, unfolding was more complex (involving an initial lag phase),and data could not be fitted to eq. 1. Instead, the time taken for thefluorescence signal to decay to 50% of the initial signal was used as anapparent t_(1/2).

From these half-times, the change in free energy of unfolding relativeto that of Termamyl could be calculated as follows:DDG=R*T*ln(t _(1/2) ^(mutant) /t _(1/2) ^(Termamyl))  (2)where R is the universal gas constant and T is the temperature (thevalue of R*T is 0.5919, giving a DDG value in kcal/mol).

By converting data to DDG values, the destabilizing/stabilizing effectsof different mutations can be compared directly and examined foradditivity (DDG₁₊₂=DDG₁+DDG₂) and synergy (DDG₁₊₂>DDG₁+DDG₂) whereDDG₁₊₂ is the energy-effect of introducing mutations 1 and 2.

Results

Unfolding of amylases at low pH and high temperature may be followed bythe decay in Trp-fluorescence. At pH 4.5 and 1 mM CaCl₂, all amylasesunfold fairly rapidly.

The unfolding data at pH 4.5 fit better to a double-exponential equationthan to a single-exponential equation. However, since the second phaseis very slow, it is approximated by a linear drift (equation 1).Unfolding at pH 6.2 and 10 micro-M CaCl₂ at 70° C. is much less rapidthan at pH 4.5 despite the low [Ca²⁺]. Unfolding is far from completewithin an hour and it is not possible to fit the data to asingle-exponential equation. Instead, the time taken for thefluorescence signal to decay to 50% of the initial signal is used as anapparent t_(1/2).

Results of the fluorescence assay are presented in Table 12.

TABLE 12 Summary of data for unfolding of Termamyl variants pH 4.5 andpH 6.2 at 70° C. pH 4.5, 1 mM CaCl₂ pH 6.2, 10 microM CaCl₂ DDG DDGMutation (kcal/mol) t_(1/2)/t_(1/2) ^(Termamyl) (kcal/mol)t_(1/2)/t_(1/2) ^(Termamyl) Wildtype 0 1.0 0   1.0 A209V −0.36 1.85−0.72 3.39 H133Y + A209V −0.77 3.67 −0.61 2.78 H156Y 0.06 0.90 −0.101.18 A181T −0.06 1.10 −0.28 1.60 A181T + A209V −0.44 2.09 <−1(73%)^(a) >5 S187D 0.37 0.54   0.19 0.72 H450Y −0.49 2.29   0.15 0.78L270D −0.35 1.8 −0.10 1.2 H156Y + A181T −0.17 1.34 −0.62 2.84 H133I−0.33 1.75 −0.42 2.02 H133Y + H156Y + A181T + A209V + −0.96 5.10 <−1(58%)^(a) >5 H293Y + A294V + G310D + H450Y V128E −0.10 1.2 −0.25 1.5H156Y + A181T + A209V −0.32 1.71 −0.49 2.30 H156Y + A181T + A209V +H450Y −0.42 2.05 −0.63 2.92 H133I + H156Y + A181T + A209V + −0.81 3.9<−1 (65%)^(a) >5 H450Y H133I + H156Y + A181T + A209V −0.70 3.3 <−1(77%)^(a) >5 Q264S −0.26 1.6 −0.14 1.3 Delta(1, 2) + L3V + M15T + R23K +S31A + −0.43 2.1 −0.82 4.0 A32E + Y33H + A35S + E36D + H37I + H156Y +A181T + A209V Q264S + N265Y −0.33 1.8 −0.07 1.1 N190F + Q264S + N265Y−1.07 6.1 −0.67 3.1 N190F + H133I + A209V + Q264S + −1.66 16.5 <−1(82%)^(a) >5 N265Y Delta(1, 2) + L3V + M15T + R23K + S31A + −0.30 1.7<−1 (66%)^(a) >5 A32E + Y33H + A35S + E36D + H37I + H156Y + A181T +N190F + A209V Delta(1, 2) + L3V + M15T + R23K + S31A + −0.43 2.1 −0.864.3 A32E + Y33H + A35S + E36D + H37I + H156Y + A181T + A209V + Q264SDelta(1, 2) + L3V + M15T + R23K + S31A + −0.36 1.8 <−1 (76%)^(a) >5A32E + Y33H + A35S + E36D + H37I + H156Y + A181T + N190F + A209V + Q264SH156Y + A181T + N190F + A209V + −1.3 8.6 <−1 (66%)^(a) >5 Q264S At pH4.5, t_(1/2) ^(Termamyl) = 200 s; at pH 6.2, t_(1/2) ^(Termamyl) = 2800s. DDG = −RT*In(t_(1/2) ^(Termamyl)/t_(1/2) ^(mutant)) ^(a)Thepercentage indicates the level to which the initial fluorescence levelhad declined in the course of 3 hours at 70° C. The slow decline isindicative of high stability.

EXAMPLE 5

Alpha-amylase Variants with Increased Specific Activity

This example summarizes the results of variants characterised by havingincreased specific activity compared to Termamyl wt. The presence ofthese substitutions either in combination with each other or as singlesubstitutions added to stabilizing substitutions increases the specificactivity of the resulting variant. The specific activity was determinedusing the alpha-amylase (Phadebas) assay described in the Materials andMethods where the activity/mg enzyme was determined. The activity wasdetermined using the following description where the pH was 7.3,temperature 37° C. and testing time 15 minutes and buffer as defined.

Specific Activity (Phadebas Assay), Index MUTATION Relative to TERMAMYLWT. S187D 260% V54I 160% BAN/Termamyl hybrid: 140% (Δ(1, 2) + L3V +M15T + R23K + S31A + A32E + Y33H + A35S + E36D + H37I) Δ(D372 + S373 +Q374) 125% Δ(K370 + G371 + D372) 125% BAN/Termamyl hybrid: 360% (Δ(1,2) + L3V + M15T + R23K + S31A + A32E + Y33H + A35S + E36D + H37I +V54I + S187D + Δ(D372 + S373 + Q374) Q264S 130% Y290E 155% Y290K 140%N190F 150%

EXAMPLE 6

Testing of Specificity Variants (saccharification)

It has been reported previously (U.S. Pat. No. 5,234,823) that, whensaccharifying with glucoamylase and pullulanase, the presence ofresidual alpha-amylase activity arising from the liquefaction process,can lead to lower yields of glucose, if the alpha-amylase is notinactivated before the saccharification stage. This inactivation can betypically carried out by adjusting the pH to below 4.3 at 95° C., beforelowering the temperature to 60° C. for saccharification.

The reason for this negative effect on glucose yield is not fullyunderstood, but it is assumed that the liquefying alpha-amylase (forexample Termamyl 120 L from B. licheniformis) generates “limit dextrins”(which are poor substrates for pullulanase), by hydrolyzing1,4-alpha-glucosidic linkages close to and on both sides of thebranching points in amylopectin. Hydrolysis of these limit dextrins byglucoamylase leads to a build up of the trisaccharide panose, which isonly slowly hydrolyzed by glucoamylase.

The development of a thermostable alpha-amylase, which does not sufferfrom this disadvantage would be a significant improvement, as noseparate inactivation step would be required.

A number of B. licheniformis alpha-amylase variants, with alteredspecificity, were evaluated by saccharifying a DE 10 Maltodextrinsubstrate with A. niger glucoamylase and B. acidopullulyticuspullulanase under conditions where the variant amylase was active.

The saccharification reactions were monitored by taking samples at 24hour intervals and analysing them by HPLC. The standard reactionconditions were:

Substrate concentration 28.2% w/w Temperature 60° C. Initial pH (at 60°C.) 4.7 Enzyme dosage Glucoamylase 0.18 AG/g DS Pullulanase 0.06 PUN/gDS alpha-amylase 60 NU/g DS

The following enzymes were used:

Glucoamylase: AMG (Novo Nordisk) 153 AG/g Pullulanase: Promozyme (NovoNordisk) 295 PUN/g Alpha-amylase: Termamyl (Novo Nordisk) 135 KNU/g V54Y313 KNU/g A52W 5,116 NU/ml D53E 3,280 NU/ml D53W 599 NU/ml A52W + V54Y134 NU/ml

The mutations listed in the alpha-amylase list above are used toindicate variants of the B. licheniformis alpha-amylase (SEQ ID NO: 2)(Termamyl) which has been modified by the indicated mutation(s).

Substrates for saccharification were prepared by dissolving 230 g DE 10spray-dried maltodextrin, prepared from common corn starch, in 460 mlboiling deionized water and adjusting the dry substance to approximately30% w/w. The pH was adjusted to 4.7 (measured at 60° C.) and aliquots ofsubstrate corresponding to 15 g dry weight, were transferred to 50 mlblue cap glass flasks.

The flasks were then placed in a shaking water bath equilibrated at 60°C., and the enzymes added. The pH was readjusted to 4.7 where necessary.2 ml samples were taken periodically, the pH adjusted to about 3.0, andthen heated in a boiling water bath for 15 minutes to inactivate theenzymes. After cooling, the samples were treated with approximately 0.1g mixed bed ion exchange resin (BIO-Rad 501 X8 (D)) for 30 minutes on arotary mixer to remove salts and soluble N. After filtration, thecarbohydrate composition was determined by HPLC. After 72 hours, thefollowing results were obtained:

Added alpha-amylase % DP₁ % DP₂ % DP₃ % DP₄₊ None (control) 96.59 2.20.3 1.0 V54Y 96.5 2.2 0.4 0.9 A52W + V54Y 96.4 2.2 0.5 0.9 Termamyl 96.32.1 0.8 0.8

Compared with the control (no active alpha-amylase present duringliquefaction), the presence of active alpha-amylase variants V54Y andA52W+V54Y did not lead to elevated panose levels (DP₃).

If these alpha-amylase variants are used for starch liquefaction, itwill not be necessary to inactivate the enzyme before the commencementof saccharification.

EXAMPLE 7

Evaluation of B. licheniformis Variants under Simulated LiquefactionConditions

The standard process for industrial starch liquefaction comprises twostages, normally referred to as primary and secondary liquefaction. Inthe first stage, a 30-40% w/w starch slurry at pH 5.5-6.0, to which hasbeen added a thermostable alpha-amylase from B. licheniformis or B.stearothermophillus, is heated to 105-110° C. in a jet cooker where livesteam is injected into the starch stream. After a holding time of 5-10minutes under pressure at this temperature, the liquefied starch isflash cooled to about 95° C. and held at that temperature for 60-120minutes.

In order to evaluate small quantities of enzyme on a laboratory scalethe following test method was used:

10 g aliquots of a suspension of common corn starch (Cerestar GL 3406)in deionized water (approx. 30% w/w) are weighed out into 100 ml conicalflasks (Schott GL 125) which are fitted with tight fitting screw caps.The pH, calcium level and enzyme dosage in the suspension can be varied.

4 flasks are used for each different set of experimental conditions. Theflasks are placed in a shaking oil-bath (Heto VS 01) maintained at 105°C. After a period of 7 minutes, cold oil is poured into the bath tolower the temperature to 95° C. For each experimental series, flasks areremoved after 20, 40, 60 and 90 minutes and immediately cooled underrunning water. One drop of 1 N HCL is added to each flask to inactivatethe enzyme. The reaction is monitored by measuring the DE (reducingsugar content expressed as glucose) using the Neocuproine method.

The details of this method can be found in “Determination of reducingsugar with improved precision. Dygert et al., 1965, Anal. Biochem. 13:368. The following DEs were recorded after 90 minutes:

pH 6.0, 5 ppm calcium added, 10.9 mg enzyme protein/g starch Hybrid +H156Y + Hybrid + H156Y + Hybrid + A181T + H156Y + A181T + H156Y +Hybrid + N190F + A181T + N190F + A181T + H156Y + N190F + H156Y + A209V +N190F + A209V + A209V + A181T + Q264S + Q264S + A181T + I.D. no.Termamyl Q264S A209V Q264S Q264S A209V Q264S N265Y N265Y A209V 045-9616.0 16.9 13.2 038-96 6.5 13.9 035-96 15.2 12.9 9.9 033-96 6.7 7.2 12.1031-96 4.5 7.0 8.8 12.5 029-96 4.0 8.7 5.2 7.7 039-96 14.9 16.3 14.4Hybrid = BAN/Termamyl PCR hybrid as described in Example 3

pH 6.0, 40 ppm calcium added, 10.9 mg enzyme protein/g starch I.D. no.Termamyl 045-96 12.6 007-97 12.1 pH 5.5, 5 ppm calcium added, 10.9 mgenzyme protein/g starch Hybrid + H156Y + Hybrid + H156Y + H156Y +A181T + A181T + N190F + A181T + N190F + N190F + A209V + I.D. no. A209V +Q264S A209V Q264S 001-97 14.8 15.2 12.6References Cited

-   Klein et al., 1992, Biochemistry 31: 8740-8746,-   Mizuno et al., 1993, J. Mol. Biol. 234: 1282-1283,-   Chang et al., 1993, J. Mol. Biol. 229: 235-238,-   Larson, 1994, J. Mol. Biol. 235: 1560-1584,-   Lawson, 1994, J. Mol. Biol. 236: 590-600,-   Qian et al., 1993, J. Mol. Biol. 231: 785-799,-   Brady et al., Acta Crystallogr. sect. B 47: 527-535,-   Swift et al., Acta Crystallogr. sect. B 47: 535-544-   A. Kadziola, Ph.D. Thesis: “An alpha-amylase from Barley and its    Complex with a Substrate Analogue Inhibitor Studied by X-ray    Crystallography”, Department of Chemistry University of Copenhagen    1993-   MacGregor, 1987, Food Hydrocolloids 1(5-6):-   Diderichsen and Christiansen, 1988, “Cloning of a maltogenic    alpha-amylase from Bacillus stearothermophilus”, FEMS Microbiol.    Letters 56: 53-60-   Hudson et al., Practical Immunology, Third edition (1989), Blackwell    Scientific Publications,-   Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,    Cold Spring Harbor, 1989-   Beaucage and Caruthers, 1981, Tetrahedron Letters 22: 1859-1869-   Matthes et al., 1984, The EMBO J. 3: 801-805.-   Saiki et al., 1988, Science 239: 487-491.-   Morinaga et al., 1984, Biotechnology 2: 646-639-   Nelson and Long, 1989, Analytical Biochemistry 180: 147-151-   Hunkapiller et al., 1984, Nature 310: 105-111-   Higuchi et al., 1988, “A general method of in vitro preparation and    specific mutagenesis of DNA fragments: study of protein and DNA    interactions”, Nucl. Acids Res. 16: 7351-7367.-   Dubnau et al., 1971, J. Mol. Biol. 56: 209-221.-   Gryczan et al., 1978, J. Bacteriol. 134: 318-329.-   S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74: 1680-1682.-   Boel et al., 1990, Biochemistry 29: 6244-6249.

The invention claimed is:
 1. A variant alpha-amylase, which (a)comprises one or more substitutions selected from the group consistingof: L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E;L270R,K,H,D,E; V283H,D; F284H; and D285N,L, wherein SEQ ID No. 2 is usedfor position numbering; (b) has at least 90% homology with the aminoacid sequence shown in SEQ ID NO: 2, 4, 6, 38, 39 or 40, and (c) hasalpha-amylase activity.
 2. The variant alpha-amylase of claim 1, whichhas at least 90% homology with the amino acid sequence shown in SEQ IDNO:
 2. 3. The variant alpha-amylase of claim 1, which has at least 95%homology with the amino acid sequence shown in SEQ ID NO:
 2. 4. Thevariant alpha-amylase of claim 1, which has at least 90% homology withthe amino acid sequence shown in SEQ ID NO:
 4. 5. The variantalpha-amylase of claim 1, which has at least 95% homology with the aminoacid sequence shown in SEQ ID NO:
 4. 6. The variant alpha-amylase ofclaim 1, which has at least 90% homology with the amino acid sequenceshown in SEQ ID NO:
 6. 7. The variant alpha-amylase of claim 1, whichhas at least 95% homology with the amino acid sequence shown in SEQ IDNO:
 6. 8. The variant alpha-amylase of claim 1, which has at least 90%homology with the amino acid sequence shown in SEQ ID NO:
 38. 9. Thevariant alpha-amylase of claim 1, which has at least 95% homology withthe amino acid sequence shown in SEQ ID NO:
 38. 10. The variantalpha-amylase of claim 1, which has at least 90% homology with the aminoacid sequence shown in SEQ ID NO:
 39. 11. The variant alpha-amylase ofclaim 1, which has at least 95% homology with the amino acid sequenceshown in SEQ ID NO:
 39. 12. The variant alpha-amylase of claim 1, whichhas at least 90% homology with the amino acid sequence shown in SEQ IDNO:
 40. 13. The variant alpha-amylase of claim 1, which has at least 95%homology with the amino acid sequence shown in SEQ ID NO:
 40. 14. Thevariant alpha-amylase of claim 1, which comprises L241R,K,D,E,F.
 15. Thevariant alpha-amylase of claim 1, which comprises A260S.
 16. The variantalpha-amylase of claim 1, which comprises W263H.
 17. The variantalpha-amylase of claim 1, which comprises Q264R,D,K,E.
 18. The variantalpha-amylase of claim 1, which comprises N265K,R,D.
 19. The variantalpha-amylase of claim 1, which comprises A269R,K,D,E.
 20. The variantalpha-amylase of claim 1, which comprises L270R,K,H,D,E.
 21. The variantalpha-amylase of claim 1, which comprises V283H,D.
 22. The variantalpha-amylase of claim 1, which comprises F284H.
 23. The variantalpha-amylase of claim 1, which comprises D285N,L.
 24. A detergentcomposition comprising an alpha-amylase variant of claim 1 and asurfactant.
 25. A method for converting starch to dextrins, comprisingtreating the starch with an alpha-amylase variant of claim 1.